Angiotensin peptides in treating marfan syndrome and related disorders

ABSTRACT

The present invention provides, among other things, methods of treating Marfan Syndrome and/or a Marfan-related disorder including administering to a subject suffering from or susceptible to Marfan Syndrome and/or a Marfan-related disorder an angiotensin (1-7) peptide. In some embodiments, the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of Marfan Syndrome and/or a Marfan-related disorder is reduced in intensity, severity, duration, or frequency or has delayed in onset.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent application61/827,271, filed May 24, 2013, the disclosure of which is herebyincorporated by reference in its entirety.

BACKGROUND

Marfan syndrome is a connective tissue disorder that affectsapproximately 1 in 5,000 individuals and is an inherited autosomaldominant disease that is caused by mutations in the gene encodingfibrillin-1 (FBN1), an extracellular matrix protein. Marfan Syndrome canmanifest in a variety of ways, often affecting one or more of oculartissue, cardiovascular tissue, and skeletal tissue. Most serious arecardiac effects, which may lead to aortic dilation and dissection,resulting in death without rapid and invasive treatment.

Prior to the present invention, there were no known effective therapiesfor Marfan Syndrome. Instead, treatment of Marfan sufferers was limitedto supportive treatment of symptoms as they develop.

SUMMARY OF THE INVENTION

The present invention provides, among other things, methods andcompositions for treating connective tissue diseases, disorders, orconditions including Marfan Syndrome and Marfan-related disorders. Thepresent invention is based, at least in part, on the realization thatangiotensin (1-7) peptides and/or angiotensin (1-7) receptor agonistsmay be used to treat one or more symptoms of Marfan Syndrome and relatedconnective tissue disorders. While angiotensin (1-7) peptides were knownto be effective for promoting angiogenesis (vascular tissue), prior tothe present invention it was unknown that angiotensin (1-7) peptidescould improve one or more symptoms of connective tissue disorders suchas Marfan Syndrome.

In some embodiments, the present invention provides methods of treatingMarfan Syndrome and/or a Marfan-related disorder including administeringto a subject suffering from or susceptible to Marfan Syndrome and/or aMarfan-related disorder an angiotensin (1-7) peptide. In someembodiments, the angiotensin (1-7) peptide is administered at aneffective dose periodically at an administration interval such that atleast one symptom or feature of Marfan Syndrome and/or a Marfan-relateddisorder is reduced in intensity, severity, duration, or frequency orhas delayed in onset. In some embodiments, the at least one symptom orfeature of Marfan Syndrome and/or a Marfan-related disorder is selectedfrom the group consisting of aortic enlargement, aortic dissection, eyelens dislocation, mitral valve prolapse, joint hypermobility, retinaldetachment, strabismus, cataracts, glaucoma, obstructive lung disease,scoliosis, temporomandibular joint disorder, dural ectasia, andosteopenia.

In addition to the treatment of Marfan Syndrome itself, the presentinvention also provides methods of treating a variety of Marfan-relateddisorders. In some embodiments, the Marfan-related disorder is selectedfrom the group consisting of: Loeys-Dietz Syndrome, Familial AorticAneurysm, Bicuspid Aortic Valve with Aortic Dilation, Familial EctopiaLentis (dislocated lens), Mitral Valve Prolapse Syndrome, MarfanHabitus, Congenital Contractural Arachnodactyly (Beals Syndrome),Stickler syndrome, Shprintzen-Goldberg syndrome, Weill-Marchesanisyndrome, and Ehlers-Danlos syndrome.

According to various embodiments, angiotensin (1-7) peptides may beadministered via any of a variety of routes. In some embodiments, theangiotensin (1-7) peptide is administered parenterally. In someembodiments, the parenteral administration is selected from intravenous,intradermal, inhalation, transdermal (topical), intraocular,intramuscular, subcutaneous, intramuscular, and/or transmucosaladministration. In some embodiments, the angiotensin (1-7) peptide isadministered orally. In some embodiments, an angiotensin (1-7) peptideis administered according to an administration interval. In someembodiments, the angiotensin (1-7) peptide is administered monthly,weekly, daily, or at variable intervals.

It is contemplated that various embodiments may use different amounts ofangiotensin (1-7) peptide. In some embodiments, the angiotensin (1-7)peptide is administered at an effective dose ranging from about 1-1,000μg/kg/day. In some embodiments, the angiotensin (1-7) peptide isadministered at an effective dose ranging from about 50-500 μg/kg/day.In some embodiments, the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 400-500 μg/kg/day. In someembodiments, the angiotensin (1-7) peptide is administered at aneffective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, or 1,000 μg/kg/day.

In some embodiments, an angiotensin (1-7) peptide may be used with oneor more medications used to treat Marfan Syndrome or Marfan-relateddisorders or one or more symptoms thereof. In some embodiments, the oneor more Marfan Syndrome and/or a Marfan-related disorder medications isselected from the group consisting of beta blockers, calcium channelblockers, ACE inhibitors, angiotensin II receptor antagonists (e.g.losartan), anticoagulants, and combinations thereof.

It is contemplated that various angiotensin (1-7) peptides may be usedin various embodiments. In some embodiments, the angiotensin (1-7)peptide comprises the naturally-occurring Angiotensin (1-7) amino acidsequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO:1).

In some embodiments, the angiotensin (1-7) peptide is a functionalequivalent of SEQ ID NO:1. In some embodiments, the functionalequivalent is a linear peptide. In some embodiments, the linear peptidecomprises a sequence that includes at least four amino acids from theseven amino acids that appear in the naturally-occurring Angiotensin(1-7), wherein the at least four amino acids maintain their relativepositions as they appear in the naturally-occurring Angiotensin (1-7).In some embodiments, the linear peptide contains 4-25 amino acids. Insome embodiments, the linear peptide is a fragment of thenaturally-occurring Angiotensin (1-7). In some embodiments, the linearpeptide contains amino acid substitutions, deletions and/or insertionsin the naturally-occurring Angiotensin (1-7). In some embodiments, thelinear peptide has an amino acid sequence ofAsp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:2).

In some embodiments, the functional equivalent is a cyclic peptide. Insome embodiments, the cyclic peptide comprises a linkage between aminoacids. In some embodiments, the linkage is located at residuescorresponding to positions Tyr⁴ and Pro⁷ in naturally-occurringAngiotensin (1-7). In some embodiments, the linkage is a thioetherbridge. In some embodiments, the cyclic peptide comprises an amino acidsequence otherwise identical to the naturally-occurring Angiotensin(1-7) amino acid sequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ IDNO:1). In some embodiments, the cyclic peptide is a 4,7-cyclizedangiotensin (1-7) with the following formula:

In some embodiments, the angiotensin (1-7) peptide comprises one or morechemical modifications to increase protease resistance, serum stabilityand/or bioavailability. In some embodiments, the one or more chemicalmodifications comprise pegylation.

In some embodiments, the present invention provides methods of treatingMarfan Syndrome and/or a Marfan-related disorder including administeringto a subject who is suffering from or susceptible to Marfan Syndromeand/or a Marfan-related disorder an angiotensin (1-7) receptor agonist.In some embodiments, the angiotensin (1-7) receptor agonist is anon-peptidic agonist. In some embodiments, the non-peptidic agonist is acompound with the following structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, theangiotensin (1-7) receptor agonist is administered orally.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows an exemplary graph of aortic root growth as determined fromechocardiogram data generated from wild-type or Fbn1^(C1039G+) miceexposed to 500 μg/kg/day TXA127 or placebo for 60 days.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification.

Agonist: As used herein, the term “agonist” refers to any molecule thathas a positive impact in a function of a protein of interest. In someembodiments, an agonist directly or indirectly enhances, strengthens,activates and/or increases an activity of a protein of interest. Inparticular embodiments, an agonist directly interacts with the proteinof interest. Such agonists can be, e.g., proteins, chemical compounds,small molecules, nucleic acids, antibodies, drugs, ligands, or otheragents.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. In particularembodiments, where a peptide is biologically active, a portion of thatpeptide that shares at least one biological activity of the peptide istypically referred to as a “biologically active” portion. In certainembodiments, a peptide has no intrinsic biological activity but thatinhibits the effects of one or more naturally-occurring angiotensincompounds is considered to be biologically active.

Carrier or diluent: As used herein, the terms “carrier” and “diluent”refers to a pharmaceutically acceptable (e.g., safe and non-toxic foradministration to a human) carrier or diluting substance useful for thepreparation of a pharmaceutical formulation. Exemplary diluents includesterile water, bacteriostatic water for injection (BWFI), a pH bufferedsolution (e.g. phosphate-buffered saline), sterile saline solution,Ringer's solution or dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic agent for thepatient to be treated. Each unit contains a predetermined quantity ofactive material calculated to produce the desired therapeutic effect. Itwill be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regimen comprises a plurality ofdoses and at least two different time periods separating individualdoses. In some embodiments, the therapeutic agent is administeredcontinuously over a predetermined period. In some embodiments, thetherapeutic agent is administered once a day (QD) or twice a day (BID).

Functional equivalent or derivative: As used herein, the term“functional equivalent” or “functional derivative” denotes a moleculethat retains a biological activity (either function or structural) thatis substantially similar to that of the original sequence. A functionalderivative or equivalent may be a natural derivative or is preparedsynthetically. Exemplary functional derivatives include amino acidsequences having substitutions, deletions, or additions of one or moreamino acids, provided that the biological activity of the protein isconserved. The substituting amino acid desirably has chemico-physicalproperties which are similar to that of the substituted amino acid.Desirable similar chemico-physical properties include, similarities incharge, bulkiness, hydrophobicity, hydrophilicity, and the like.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form ofdisease as the individual being treated, who is about the same age asthe individual being treated (to ensure that the stages of the diseasein the treated individual and the control individual(s) are comparable).

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Prevent: As used herein, the term “prevent” or “prevention”, when usedin connection with the occurrence of a disease, disorder, and/orcondition, refers to reducing the risk of developing the disease,disorder and/or condition. See the definition of “risk.”

Polypeptide: The term “polypeptide” as used herein refers a sequentialchain of amino acids linked together via peptide bonds. The term is usedto refer to an amino acid chain of any length, but one of ordinary skillin the art will understand that the term is not limited to lengthychains and can refer to a minimal chain comprising two amino acidslinked together via a peptide bond. As is known to those skilled in theart, polypeptides may be processed and/or modified.

Protein: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does not require permanent ortemporary physical association with other polypeptides in order to formthe discrete functioning unit, the terms “polypeptide” and “protein” maybe used interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically associate with one another,the term “protein” refers to the multiple polypeptides that arephysically coupled and function together as the discrete unit.

Risk: As will be understood from context, a “risk” of a disease,disorder, and/or condition comprises a likelihood that a particularindividual will develop a disease, disorder, and/or condition (e.g.,Marfan Syndrome or a Marfan-related disorder). In some embodiments, riskis expressed as a percentage. In some embodiments, risk is from 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. Insome embodiments risk is expressed as a risk relative to a riskassociated with a reference sample or group of reference samples. Insome embodiments, a reference sample or group of reference samples havea known risk of a disease, disorder, condition and/or event (e.g.,Marfan Syndrome or a Marfan-related disorder). In some embodiments areference sample or group of reference samples are from individualscomparable to a particular individual. In some embodiments, relativerisk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent to maintain its therapeutic efficacy (e.g., all orthe majority of its intended biological activity and/or physiochemicalintegrity) over extended periods of time. The stability of a therapeuticagent, and the capability of the pharmaceutical composition to maintainstability of such therapeutic agent, may be assessed over extendedperiods of time (e.g., for at least 1, 3, 6, 12, 18, 24, 30, 36 monthsor more). In certain embodiments, pharmaceutical compositions describedherein have been formulated such that they are capable of stabilizing,or alternatively slowing or preventing the degradation, of one or moretherapeutic agents formulated therewith. In the context of a formulationa stable formulation is one in which the therapeutic agent thereinessentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization).

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre and post natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,condition, or event (for example, Muscular Dystrophy) may becharacterized by one or more of the following: (1) a genetic mutationassociated with development of the disease, disorder, and/or condition;(2) a genetic polymorphism associated with development of the disease,disorder, and/or condition; (3) increased and/or decreased expressionand/or activity of a protein associated with the disease, disorder,and/or condition; (4) habits and/or lifestyles associated withdevelopment of the disease, disorder, condition, and/or event (5) havingundergone, planning to undergo, or requiring a transplant. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will develop the disease, disorder, and/or condition.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition will not develop the disease, disorder,and/or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, improvedcompositions and methods for treating or reducing the risk of connectivetissue diseases, disorders and/or conditions such as Marfan Syndrome andMarfan-related disorders.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise.

Marfan Syndrome

Marfan Syndrome is a systemic connective tissue disorder resulting fromone or more mutations in the fibrillin-1 (FBN-1) gene. Such mutationsresult in a variable constellation of symptoms with at least one thingin common, expansion of one or more tissues beyond normal limits. Forexample, one of the most serious symptoms of Marfan Syndrome is aorticdilation and is caused by stretching of the cardiac tissue to the pointthat mechanical stability and or mechanical compliance is compromised,potentially leading to failure such as aortic dissection.

Marfan Syndrome is largely an inherited condition and is autosomaldominant, though it is suspected that approximately 25% of Marfansufferers manifest the disease as a result of spontaneous mutation. Itis suspected that dysregulation of FBN1 results in increased TGF-βsignaling.

Many Marfan sufferers exhibit a constellation of skeletal symptoms knownas Marfanoid Habitus (Marfan body type, described below). In addition toMarfanoid Habitus or similar symptomatic presentation, Marfan sufferersmay exhibit a wide array of other symptoms including, but not limited toaortic enlargement, aortic dissection, eye lens dislocation, mitralvalve prolapse, retinal detachment, strabismus, cataracts, glaucoma,obstructive lung disease, scoliosis, temporomandibular joint disorder,dural ectasia, and osteopenia.

Current treatments for Marfan sufferers are generally limited tosupportive treatments, including treatment of symptoms as they arise. Asan example of symptomatic treatments, development of cardiacabnormalities caused by Marfan Syndrome are typically treated with betablockers to cause the heart to beat slower and with less force in orderto slow the progression of aortic dilation. Once aortic dilation hasprogressed, surgery is often used to replace the dilated segment beforeit tears as tearing can lead to death in minutes. Without wishing to beheld to a particular theory, it is thought that vascular pathology inMarfan Syndrome is associated with changes in connective tissuearchitecture manifested by reduced mechanical compliance, abnormalextracellular matrix modeling, and progressive aneurysm development thatultimately lead to aortic wall degradation.

Marfanoid Habitus

Marfanoid Habitus is a Marfan-related disorder that typically includesthe symptoms of long limbs, arachnodactyly, and hyperlaxity of joints(i.e. joint hypermobility). Other symptoms common among MarfanoidHabitus sufferers include scoliosis, chest wall abnormalities, flatfeet, and a highly arched roof of the mouth, though others may beexhibited as well. Sufferers of Marfanoid Habitus tend not to manifestthe aortic and ocular issues common among Marfan sufferers. In general,the skeletal issues comprising Marfanoid Habitus are notlife-threatening, but do cause significant discomfort and evendisability in affected individuals.

Loeys-Dietz Syndrome

Loeys-Dietz Syndrome is an autosomal dominant genetic disorder whichshares may features with Marfan Syndrome, including scoliosis, jointhypermobility, and congenital heart problems. However, the genetic basisof Loeys-Dietz Syndrome is one or more mutations in the genes encodingtransforming growth factor beta receptor-1 or -2 (TGFBR1 or TGFBR2,respectively), rather than a mutation in the FBN1 gene as in MarfanSyndrome.

Sufferers of Loeys-Dietz Syndrome may exhibit symptoms distinct fromMarfan sufferers. These symptoms include but are not limited to widelyspaced eyes, cleft palate, club foot and translucency of the skin. Aswith Marfan Syndrome, there are no known cures for the disorder, andcardiac abnormalities, including aortic aneurysm and dissection are themost serious effects of Loeys-Dietz.

Congenital Contractural Arachnodactyly (Beals Syndrome)

Beals Syndrome is a Marfan-Related disorder having as its genetic basisone or more mutations in the fibrillin-2 (FBN-2) gene, as opposed toFBN-1 mutation(s) underlying Marfan Syndrome. Beals Syndrome suffererstypically experience contractures at birth, with the degree ranging frommild contracture of large joints such as the elbow and/or knee, to largecontractures resulting in scoliosis and sometimes even kyphosis. Severecontractures are typically treated via surgery.

Sufferers of Beals Syndrome typically have long, thin fingers and toeswith contractures limiting or even preventing straightening of digits.Also, sufferers often have unusual ears that appear crumpled and mayexperience joint dislocation, often in the knee (patellar dislocation).In some cases, contractures may be present from birth and reduce inseverity over time, though some sufferers do not experience significantreductions in contracture. In some cases, contractures may be so severeas to fracture long bones, though this is not typical.

Beals Syndrome may be diagnosed through observation of crumpled ears andother congenital contractures, something not typically observed inMarfan sufferers. Additionally, Beals sufferers do not usually manifestthe ocular and cardiovascular complications seen in Marfan sufferers.

Stickler Syndrome

Stickler Syndrome is a Marfan-Related disorder of the connective tissue,specifically collagen. Stickler syndrome is most often characterized bydistinctive facial abnormalities, ocular problems, and joint problems.Unlike Marfan Syndrome, it appears that more than one gene may beaffected in Stickler sufferers. Mutations in the COL2A1, COL11A1,COL11A2, and COL9A1 genes have all been reported, with mutation(s) inCOL2A1 being most common (found in ˜75% of Stickler sufferers).

A characteristic feature of Stickler Syndrome is a flattened facialappearance. This is thought to be caused by underdeveloped bones in themiddle of the face, including the cheekbones and bridge of the nose. Itis also common for Stickler sufferers to exhibit a constellation ofsymptoms known as the Pierre Robin sequence. The Pierre Robin sequenceincludes a U-shaped or even V-shaped cleft palate, a small lower jaw,and a tongue too large for the space formed by the small lower jaw. Itis somewhat common for Stickler sufferers to have an obstructed airwaydue to the Pierre Robin sequence.

The most common treatment employed on behalf of Stickler sufferers issurgery to correct maxillofacial defects and clear obstructed airways.In addition, pain medications are sometimes required to alleviatediscomfort due to malformed anatomy.

Shprintzen-Goldberg Syndrome

Shprintzen-Goldberg Syndrome (SGS) is an extremely rare multiple anomalysyndrome that typically includes craniosynostosis, multiple abdominalhernias, cognitive impairment, and other skeletal malformations. Thereis uncertainty about the cause of the disorder with some reports linkingdevelopment to the FBN1 gene, and others finding no such causal link.

In contrast to Marfan Syndrome, SGS sufferers do exhibit cognitivedefects and brain anomalies such as hydrocephalus and dilation of thelateral ventricles. Diagnosis is at times complicated by the fact thatSGS sufferers may also exhibit cardiovascular anomalies including mitralvalve prolapse, aortic regurgitation, and skeletal abnormalitiesincluding arachnodactyly, scoliosis, joint hypermobility, sometimesresulting in difficulty in diagnosing over Marfan Syndrome or otherMarfan-related disorders.

As with other Marfan-related disorders, treatment of SGS is limited tosymptomatic treatments including surgery, physiotherapy, and painmanagement.

Weill-Marchesani Syndrome

Weill-Marchesani Syndrome (WMS) is a Marfan-related connective tissuedisorder that is characterized by short stature, broad head and otherface and hand abnormalities including short fingers and small, sphericallenses of the eye that are particularly susceptible to dislocation. Theaverage height of male sufferers is between four feet eight inches tofive feet six inches, while female suffers tend to range between fourfeet three inches to five feet two inches.

While WMS sufferers may have mutations in their FBN1 gene, mutations inthe ADAMTS10 gene are also common, and there have been cases where WMSsufferers had no mutation in either their FBN1 or ADAMTS10 genes.Reported cases have had both autosomal dominant and autosomal recessivemanifestations. As a result of this genetic variability, the cause ofWMS is poorly understood. As with several Marfan-related disorders,treatment is mostly supportive in nature and there is no known cure.

Ehlers-Danlos Syndrome

Ehlers-Danlos Syndrome is a Marfan-related connective tissue disordercharacterized by one or more defects in the synthesis of collagen (typeI or III). The severity of the defect leads to a wide range of symptomsthat can be mild or life-threatening, depending upon the specificclinical manifestation. The symptoms are similar to those found in otherMArfan-related syndromes including hyper flexible joints, scoliosis, andvarious cardiovascular abnormalities. As with other Marfan-relateddisorders, there is no cure and treatments are supportive in nature.

Unlike Marfan Syndrome, Ehlers-Danlos sufferers may also exhibitassorted skin-related conditions including fragile skin that tearseasily, abnormal wound healing, and fatty growths on forearms and/orshins. The range of symptoms exhibited by Ehlers-Danlos sufferers may bedue to the wide range of genetic abnormalities that may play a role indevelopment of the disorder including COL1A1, COL1A2, COL3A1, COL5A1,COL5A2, and TNXB. Mutation of these genes is known to affect one or moreof the structure, processing or production of proper collagen.

Familial Aortic Aneurysm

Familial Aortic Aneurysm is a Marfan-related disorder that oftenmanifests in Marfan sufferers. It is an autosomal dominant disorder oflarge arteries. Familial aortic aneurysm is caused by a breakdown ofcollagen, elastin and smooth muscle that can be caused by normal agingin addition to genetic abnormalities such as that underlying MarfanSyndrome. Aortic aneurysm results in a significantly increased risk ofaortic dissection, which can quickly lead to death.

Bicuspid Aortic Valve with Aortic Dilation

A bicuspid aortic valve is a condition wherein two of the aorticvalvular leaflets fuse, resulting in a bicuspid valve, as opposed to thenormal tricuspid aortic valve. Complications arising from bicuspidaortic valve range from a heart murmur to aortic stenosis or even aorticregurgitation. Bicuspid valve is a condition often associated withMarfan Syndrome and, as such, it is herein considered a Marfan-relateddisorder.

Ectopia Lentis (Dislocated Lens)

Ectopia lentis is a Marfan-related disorder wherein the eye'scrystalline lens is displaced from its normal location. Ectopia lentismay have a variety of causes, but it is inherited at a higher frequencyamong Marfan sufferers. A partial dislocation may also be referred to aslens subluxation while a complete dislocation may also be referred to aslens luxation. Ectopia lentis may result in several difficultiesincluding nearsightedness, astigmatism (irregular curvature of the eye),and fluctuated or blurred vision.

Mitral Valve Prolapse Syndrome

Mitral valve prolapse is characterized by the displacement of anabnormally thickened mitral valve leaflet into the left atrium duringsystole. While mitral valve prolapse may vary significantly in severity,severe forms may result in mitral regurgitation, congestive heartfailure, and even cardiac arrest. For reasons that are not yet clear,mitral valve prolapse is more common in Marfan sufferers than in thegeneral population, this it is herein considered a Marfan-relateddisorder.

Angiotensin (1-7) Peptides

As used herein, the term “angiotensin (1-7) peptide” refers to bothnaturally-occurring Angiotensin (1-7) and any functional equivalent,analogue or derivative of naturally-occurring Angiotensin (1-7). As usedherein, “peptide” and “polypeptide” are interchangeable terms and referto two or more amino acids bound together by a peptide bond. As usedherein, the terms “peptide” and “polypeptide” include both linear andcyclic peptide. The terms “angiotensin-(1-7)”, “Angiotensin-(1-7)”, and“Ang-(1-7)” are used interchangeably.

Naturally-Occurring Angiotensin (1-7)

Naturally-occurring Angiotensin (1-7) (also referred to as Ang-(1-7)) isa seven amino acid peptide shown below:

(SEQ ID NO: 1) Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ 

It is part of the renin-angiotensin system and is converted from aprecursor, also known as Angiotensinogen, which is an α-2-globulin thatis produced constitutively and released into the circulation mainly bythe liver. Angiotensinogen is a member of the serpin family and alsoknown as renin substrate. Human angiotensinogen is 452 amino acids long,but other species have angiotensinogen of varying sizes. Typically, thefirst 12 amino acids are the most important for angiotensin activity:

(SEQ ID NO: 3) Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹-Leu¹⁰-Val¹¹-Ile¹²

Different types of angiotensin may be formed by the action of variousenzymes. For example, Angiotensin (1-7) is generated by action ofAngiotensin-converting enzyme 2 (ACE 2).

Ang-(1-7) is an endogenous ligand for Mas receptors. Mas receptors areG-protein coupled receptor containing seven transmembrane spanningregions. As used herein, the term “angiotensin-(1-7) receptor’encompasses the G Protein-Coupled Mas Receptors.

As used herein, the term “naturally-occurring Angiotensin (1-7)”includes any Angiotensin (1-7) peptide purified from natural sources andany recombinantly produced or chemically synthesized peptides that havean amino acid sequence identical to that of the naturally-occurringAngiotensin (1-7).

Functional Equivalents, Analogs or Derivatives of Ang-(1-7)

In some embodiments, an angiotensin (1-7) peptide suitable for thepresent invention is a functional equivalent of naturally-occurringAng-(1-7). As used herein, a functional equivalent ofnaturally-occurring Ang-(1-7) refers to any peptide that shares aminoacid sequence identity to the naturally-occurring Ang-(1-7) and retainsubstantially the same or similar activity as the naturally-occurringAng-(1-7). For example, in some embodiments, a functional equivalent ofnaturally-occurring Ang-(1-7) described herein has pro-angiogenicactivity as determined using methods described herein or known in theart, or an activity such as nitric oxide release, vasodilation, improvedendothelial function, antidiuresis, or one of the other propertiesdiscussed herein, that positively impacts angiogenesis. In someembodiments, a functional equivalent of naturally-occurring Ang-(1-7)described herein can bind to or activate an angiotensin-(1-7) receptor(e.g., the G protein-coupled Mas receptor) as determined using variousassays described herein or known in the art. In some embodiments, afunctional equivalent of Ang-(1-7) is also referred to as an angiotensin(1-7) analogue or derivative, or functional derivative.

Typically, a functional equivalent of angiotensin (1-7) shares aminoacid sequence similarity to the naturally-occurring Ang-(1-7). In someembodiments, a functional equivalent of Ang-(1-7) according to theinvention contains a sequence that includes at least 3 (e.g., at least4, at least 5, at least 6, at least 7) amino acids from the seven aminoacids that appear in the naturally-occurring Ang-(1-7), wherein the atleast 3 (e.g., at least 4, at least 5, at least 6, or at least 7) aminoacids maintain their relative positions and/or spacing as they appear inthe naturally-occurring Ang-(1-7).

In some embodiments, a functional equivalent of angiotensin (1-7) alsoencompass any peptide that contain a sequence at least 50% (e.g., atleast 60%, 70%, 80%, or 90%) identical to the amino acid sequence ofnaturally-occurring Ang-(1-7). Percentage of amino acid sequenceidentity can be determined by alignment of amino acid sequences.Alignment of amino acid sequences can be achieved in various ways thatare within the skill in the art, for instance, using publicly availablecomputer software such as BLAST, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.Preferably, the WU-BLAST-2 software is used to determine amino acidsequence identity (Altschul et al., Methods in Enzymology 266, 460-480(1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 usesseveral search parameters, most of which are set to the default values.The adjustable parameters are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11. HSP score (S) andHSP S2 parameters are dynamic values and are established by the programitself, depending upon the composition of the particular sequence,however, the minimum values may be adjusted and are set as indicatedabove.

In some embodiments, a functional equivalent, analogue or derivative ofAng-(1-7) is a fragment of the naturally-occurring Ang-(1-7). In someembodiments, a functional equivalent, analogue or derivative ofAng-(1-7) contains amino acid substitutions, deletions and/or insertionsin the naturally-occurring Ang-(1-7). Ang-(1-7) functional equivalents,analogues or derivatives can be made by altering the amino acidsequences by substitutions, additions, and/or deletions. For example,one or more amino acid residues within the sequence of thenaturally-occurring Ang-(1-7) (SEQ ID NO:1) can be substituted byanother amino acid of a similar polarity, which acts as a functionalequivalent, resulting in a silent alteration. Substitution for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the positively charged(basic) amino acids include arginine, lysine, and histidine. Thenonpolar (hydrophobic) amino acids include leucine, isoleucine, alanine,phenylalanine, valine, proline, tryptophane, and methionine. Theuncharged polar amino acids include serine, threonine, cysteine,tyrosine, asparagine, and glutamine. The negatively charged (acid) aminoacids include glutamic acid and aspartic acid. The amino acid glycinemay be included in either the nonpolar amino acid family or theuncharged (neutral) polar amino acid family. Substitutions made within afamily of amino acids are generally understood to be conservativesubstitutions. For example, the amino acid sequence of a peptideinhibitor can be modified or substituted.

Examples of Ang-(1-7) functional equivalents, analogues and derivativesare described in the section entitled “Exemplary Angiotensin(1-7)Peptides” below.

An angiotensin-(1-7) peptide can be of any length. In some embodiments,an angiotensin-(1-7) peptide according to the present invention cancontain, for example, from 4-25 amino acids (e.g., 4-20, 4-15, 4-14,4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7 amino acids). In some embodiments,the linear peptide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids.

In some embodiments, an angiotensin-(1-7) peptide contains one or moremodifications to increase protease resistance, serum stability and/orbioavailability. In some embodiments, suitable modifications areselected from pegylation, acetylation, glycosylation, biotinylation,substitution with D-amino acid and/or un-natural amino acid, and/orcyclization of the peptide.

As used herein, the term “amino acid,” in its broadest sense, refers toany compound and/or substance that can be incorporated into apolypeptide chain. In certain embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In certain embodiments, an amino acid is anaturally-occurring amino acid. In certain embodiments, an amino acid isa synthetic or un-natural amino acid (e.g., α,α-disubstituted aminoacids, N-alkyl amino acids); in some embodiments, an amino acid is ad-amino acid; in certain embodiments, an amino acid is an 1-amino acid.“Standard amino acid” refers to any of the twenty standard amino acidscommonly found in naturally occurring peptides including both l- andd-amino acids which are both incorporated in peptides in nature.“Nonstandard” or “unconventional amino acid” refers to any amino acid,other than the standard amino acids, regardless of whether it isprepared synthetically or obtained from a natural source. As usedherein, “synthetic or un-natural amino acid” encompasses chemicallymodified amino acids, including but not limited to salts, amino acidderivatives (such as amides), and/or substitutions. Amino acids,including carboxy- and/or amino-terminal amino acids in peptides, can bemodified by methylation, amidation, acetylation, and/or substitutionwith other chemical groups that can change the peptide's circulatinghalf-life without adversely affecting its activity. Examples ofunconventional or un-natural amino acids include, but are not limitedto, citrulline, ornithine, norleucine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl-leucine(MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (MeAla).Amino acids may participate in a disulfide bond. The term “amino acid”is used interchangeably with “amino acid residue,” and may refer to afree amino acid and/or to an amino acid residue of a peptide. It will beapparent from the context in which the term is used whether it refers toa free amino acid or a residue of a peptide.

In certain embodiments, angiotensin-(1-7) peptides contain one or moreL-amino acids, D-amino acids, and/or un-natural amino acids.

In addition to peptides containing only naturally occurring amino acids,peptidomimetics or peptide analogs are also encompassed by the presentinvention. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. The non-peptide compounds are termed “peptidemimetics” or peptidomimetics (Fauchere et al., Infect. Immun. 54:283-287(1986); Evans et al., J. Med. Chem. 30:1229-1239 (1987)). Peptidemimetics that are structurally related to therapeutically usefulpeptides and may be used to produce an equivalent or enhancedtherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to the paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity) such asnaturally-occurring receptor-binding polypeptides, but have one or morepeptide linkages optionally replaced by linkages such as —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH— (cis and trans), —CH₂SO—, —CH(OH)CH₂—,—COCH₂—etc., by methods well known in the art (Spatola, Peptide BackboneModifications, Vega Data, 1(3):267 (1983); Spatola et al. Life Sci.38:1243-1249 (1986); Hudson et al. Int. J. Pept. Res. 14:177-185 (1979);and Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids,Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York,). Suchpeptide mimetics may have significant advantages overnaturally-occurring polypeptides including more economical production,greater chemical stability, enhanced pharmacological properties (e.g.,half-life, absorption, potency, efficiency, etc.), reduced antigenicityand others.

Ang-(1-7) peptides also include other types of peptide derivativescontaining additional chemical moieties not normally part of thepeptide, provided that the derivative retains the desired functionalactivity of the peptide. Examples of such derivatives include (1) N-acylderivatives of the amino terminal or of another free amino group,wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl,octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such asF-moc (fluorenylmethyl-O—CO—); (2) esters of the carboxy terminal or ofanother free carboxy or hydroxyl group; (3) amide of thecarboxy-terminal or of another free carboxyl group produced by reactionwith ammonia or with a suitable amine; (4) phosphorylated derivatives;(5) derivatives conjugated to an antibody or other biological ligand andother types of derivatives; and (6) derivatives conjugated to apolyethylene glycol (PEG) chain.

Ang-(1-7) peptides may be obtained by any method of peptide synthesisknown to those skilled in the art, including synthetic (e.g., exclusivesolid phase synthesis, partial solid phase synthesis, fragmentcondensation, classical solution synthesis, native-chemical ligation)and recombinant techniques. For example, the peptides or peptidesderivatives can be obtained by solid phase peptide synthesis, which inbrief, consist of coupling the carboxyl group of the C-terminal aminoacid to a resin (e.g., benzhydrylamine resin, chloromethylated resin,hydroxymethyl resin) and successively adding N-alpha protected aminoacids. The protecting groups may be any such groups known in the art.Before each new amino acid is added to the growing chain, the protectinggroup of the previous amino acid added to the chain is removed. Suchsolid phase synthesis has been disclosed, for example, by Merrifield, J.Am. Chem. Soc. 85: 2149 (1964); Vale et al., Science 213:1394-1397(1981), in U.S. Pat. Nos. 4,305,872 and 4,316,891, Bodonsky et al. Chem.Ind. (London), 38:1597 (1966); and Pietta and Marshall, Chem. Comm. 650(1970) by techniques reviewed in Lubell et al. “Peptides” Science ofSynthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809 (2005).The coupling of amino acids to appropriate resins is also well known inthe art and has been disclosed in U.S. Pat. No. 4,244,946. (Reviewed inHouver-Weyl, Methods of Organic Chemistry. Vol E22a. Synthesis ofPeptides and Peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme.Stuttgart. New York 2002).

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures of cell cultures, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as, for example,Ausubel et al., Current Protocols in Molecular Biology, WileyInterscience, New York, 2001; and Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press,N.Y., 2001.

During any process of the preparation of an Ang-(1-7) peptide, it may bedesirable to protect sensitive reactive groups on any of the moleculeconcerned. This may be achieved by means of conventional protectinggroups such as those described in Protective Groups In Organic Synthesisby T.W. Greene & P.G.M. Wuts, 1991, John Wiley and Sons, New-York; andPeptides: chemistry and Biology by Sewald and Jakubke, 2002, Wiley-VCH,Wheinheim p. 142. For example, alpha amino protecting groups includeacyl type protecting groups (e.g., trifluoroacetyl, formyl, acetyl),aliphatic urethane protecting groups (e.g., t-butyloxycarbonyl (BOC),cyclohexyloxycarbonyl), aromatic urethane type protecting groups (e.g.,fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbzderivatives) and alkyl type protecting groups (e.g., triphenyl methyl,benzyl). The amino acids side chain protecting groups include benzyl(for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl,cyclohexyl (Asp, His), Boc (Arg, His, Cys) etc. The protecting groupsmay be removed at a convenient subsequent stage using methods known inthe art.

Further, Ang-(1-7) peptides may be synthesized according to the FMOCprotocol in an organic phase with protective groups. Desirably, thepeptides are purified with a yield of 70% with high-pressure liquidchromatography (HPLC) on a C18 chromatography column and eluted with anacetonitrile gradient of 10-60%. The molecular weight of a peptide canbe verified by mass spectrometry (reviewed in Fields, G. B. “Solid-PhasePeptide Synthesis” Methods in Enzymology. Vol. 289, Academic Press,1997).

Alternatively, Ang-(1-7) peptides may be prepared in recombinant systemsusing, for example, polynucleotide sequences encoding the polypeptides.It is understood that a polypeptide may contain more than one of theabove-described modifications within the same polypeptide.

While peptides may be effective in eliciting a biological activity invitro, their effectiveness in vivo might be reduced by the presence ofproteases. Serum proteases have specific substrate requirements. Thesubstrate must have both L-amino acids and peptide bonds for cleavage.Furthermore, exopeptidases, which represent the most prominent componentof the protease activity in serum, usually act on the first peptide bondof the peptide and require a free N-terminus (Powell et al., Pharm. Res.10:1268-1273 (1993)). In light of this, it is often advantageous to usemodified versions of peptides. The modified peptides retain thestructural characteristics of the original L-amino acid peptides thatconfer the desired biological activity of Ang-(1-7) but areadvantageously not readily susceptible to cleavage by protease and/orexopeptidases.

Systematic substitution of one or more amino acids of a consensussequence with D-amino acid of the same type (e.g., D-lysine in place ofL-lysine) may be used to generate more stable peptides. Thus, a peptidederivative or peptidomimetic of the present invention may be all L, allD or mixed D, L peptide, in either forward or reverse order. Thepresence of an N-terminal or C-terminal D-amino acid increases the invivo stability of a peptide since peptidases cannot utilize a D-aminoacid as a substrate (Powell et al., Pharm. Res. 10:1268-1273 (1993)).Reverse-D peptides are peptides containing D-amino acids, arranged in areverse sequence relative to a peptide containing L-amino acids. Thus,the C-terminal residue of an L-amino acid peptide becomes N-terminal forthe D-amino acid peptide, and so forth. Reverse D-peptides retain thesame secondary conformation and therefore similar activity, as theL-amino acid peptides, but are more resistant to enzymatic degradationin vitro and in vivo, and thus can have greater therapeutic efficacythan the original peptide (Brady and Dodson, Nature 368:692-693 (1994);Jameson et al., Nature 368:744-746 (1994)). Similarly, a reverse-Lpeptide may be generated using standard methods where the C-terminus ofthe parent peptide becomes takes the place of the N-terminus of thereverse-L peptide. It is contemplated that reverse L-peptides of L-aminoacid peptides that do not have significant secondary structure (e.g.,short peptides) retain the same spacing and conformation of the sidechains of the L-amino acid peptide and therefore often have the similaractivity as the original L-amino acid peptide. Moreover, a reversepeptide may contain a combination of L- and D-amino acids. The spacingbetween amino acids and the conformation of the side chains may beretained resulting in similar activity as the original L-amino acidpeptide.

Another effective approach to confer resistance to peptidases acting onthe N-terminal or C-terminal residues of a peptide is to add chemicalgroups at the peptide termini, such that the modified peptide is nolonger a substrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum (Powellet al., Pharm. Res. 10:1268-1273 (1993)). Other chemical modificationswhich enhance serum stability include, but are not limited to, theaddition of an N-terminal alkyl group, consisting of a lower alkyl offrom one to twenty carbons, such as an acetyl group, and/or the additionof a C-terminal amide or substituted amide group. In particular, thepresent invention includes modified peptides consisting of peptidesbearing an N-terminal acetyl group and/or a C-terminal amide group.

Substitution of non-naturally-occurring amino acids for natural aminoacids in a subsequence of the peptides can also confer resistance toproteolysis. Such a substitution can, for instance, confer resistance toproteolysis by exopeptidases acting on the N-terminus without affectingbiological activity. Examples of non-naturally-occurring amino acidsinclude α,α-disubstituted amino acids, N-alkyl amino acids, C-α-methylamino acids, β-amino acids, and β-methyl amino acids. Amino acidsanalogs useful in the present invention may include, but are not limitedto, β-alanine, norvaline, norleucine, 4-aminobutyric acid, orithine,hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine,2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine,phenylglycine, o-phosphoserine, N-acetyl serine, N-formylmethionine,3-methylhistidine and other unconventional amino acids. Furthermore, thesynthesis of peptides with non-naturally-occurring amino acids isroutine in the art.

In addition, constrained peptides comprising a consensus sequence or asubstantially identical consensus sequence variation may be generated bymethods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem.61:387-418 (1992)). For example, constrained peptides may be generatedby adding cysteine residues capable of forming disulfide bridges and,thereby, resulting in a cyclic peptide. Cyclic peptides can beconstructed to have no free N- or C-termini. Accordingly, they are notsusceptible to proteolysis by exopeptidases, although they may besusceptible to endopeptidases, which do not cleave at peptide termini.The amino acid sequences of the peptides with N-terminal or C-terminalD-amino acids and of the cyclic peptides are usually identical to thesequences of the peptides to which they correspond, except for thepresence of N-terminal or C-terminal D-amino acid residue, or theircircular structure, respectively.

Cyclic Peptides

In some embodiments, a functional equivalent, analogue or derivative ofnaturally-occurring Ang-(1-7) is a cyclic peptide. As used herein, acyclic peptide has an intramolecular covalent bond between twonon-adjacent residues. The intramolecular bond may be a backbone tobackbone, side-chain to backbone or side-chain to side-chain bond (i.e.,terminal functional groups of a linear peptide and/or side-chainfunctional groups of a terminal or interior residue may be linked toachieve cyclization). Typical intramolecular bonds include disulfide,amide and thioether bonds. A variety of means for cyclizing polypeptidesare well known in the art, as are many other modifications that can bemade to such peptides. For a general discussion, see InternationalPatent Publication Nos. WO 01/53331 and WO 98/02452, the contents ofwhich are incorporated herein by reference. Such cyclic bonds and othermodifications can also be applied to the cyclic peptides and derivativecompounds of this invention.

Cyclic peptides as described herein may comprise residues of L-aminoacids, D-amino acids, or any combination thereof. Amino acids may befrom natural or non-natural sources, provided that at least one aminogroup and at least one carboxyl group are present in the molecule; α-and β-amino acids are generally preferred. Cyclic peptides may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonylation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzylation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Suitable derivatives include amino acids having an N-acetyl group(such that the amino group that represents the N-terminus of the linearpeptide prior to cyclization is acetylated) and/or a C-terminal amidegroup (i.e., the carboxy terminus of the linear peptide prior tocyclization is amidated). Residues other than common amino acids thatmay be present with a cyclic peptide include, but are not limited to,penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine,β-mercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, α-aminoadipic acid, m-aminomethylbenzoic acid andα,β-diaminopropionic acid.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved by any of a variety oftechniques well known in the art. Within one embodiment, a bond may begenerated between reactive amino acid side chains. For example, adisulfide bridge may be formed from a linear peptide comprising twothiol-containing residues by oxidizing the peptide using any of avariety of methods. Within one such method, air oxidation of thiols cangenerate disulfide linkages over a period of several days using eitherbasic or neutral aqueous media. The peptide is used in high dilution tominimize aggregation and intermolecular side reactions. Alternatively,strong oxidizing agents such as I₂ and K₃Fe(CN)₆ can be used to formdisulfide linkages. Those of ordinary skill in the art will recognizethat care must be taken not to oxidize the sensitive side chains of Met,Tyr, Trp or His. Within further embodiments, cyclization may be achievedby amide bond formation. For example, a peptide bond may be formedbetween terminal functional groups (i.e., the amino and carboxy terminiof a linear peptide prior to cyclization). Within another suchembodiment, the linear peptide comprises a D-amino acid. Alternatively,cyclization may be accomplished by linking one terminus and a residueside chain or using two side chains, with or without an N-terminalacetyl group and/or a C-terminal amide. Residues capable of forming alactam bond include lysine, ornithine (Orn), α-amino adipic acid,m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate. Methods for forming amide bonds are generally well known inthe art. Within one such method, carbodiimide-mediated lactam formationcan be accomplished by reaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resulting in the formation of an O-acylurea that can bereacted immediately with the free amino group to complete thecyclization. Alternatively, cyclization can be performed using the azidemethod, in which a reactive azide intermediate is generated from analkyl ester via a hydrazide. Alternatively, cyclization can beaccomplished using activated esters. The presence of electronwithdrawing substituents on the alkoxy carbon of esters increases theirsusceptibility to aminolysis. The high reactivity of esters ofp-nitrophenol, N-hydroxy compounds and polyhalogenated phenols has madethese “active esters” useful in the synthesis of amide bonds. Within afurther embodiment, a thioether linkage may be formed between the sidechain of a thiol-containing residue and an appropriately derivatizedα-amino acid. By way of example, a lysine side chain can be coupled tobromoacetic acid through the carbodiimide coupling method (DCC, EDAC)and then reacted with the side chain of any of the thiol containingresidues mentioned above to form a thioether linkage. In order to formdithioethers, any two thiol containing side-chains can be reacted withdibromoethane and diisopropylamine in DMF.

Exemplary Angiotensin-(1-7) Peptides

In certain aspects, the invention provides linear angiotensin-(1-7)peptides. As discussed above, the structure of naturally-occurringAng-(1-7) is as follows:

(SEQ ID NO: 1) Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ 

The peptides and peptide analogs of the invention can be generallyrepresented by the following sequence:

(SEQ ID NO: 4) Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷,or a pharmaceutically acceptable salt thereof.

Xaa¹ is any amino acid or a dicarboxylic acid. In certain embodiments,Xaa¹ is Asp, Glu, Asn, Acpc (1-aminocyclopentane carboxylic acid), Ala,Me₂Gly (N,N-dimethylglycine), Pro, Bet (betaine,1-carboxy-N,N,N-trimethylmethanaminium hydroxide), Glu, Gly, Asp, Sar(sarcosine) or Suc (succinic acid). In certain such embodiments, Xaa¹ isa negatively-charged amino acid, such as Asp or Glu, typically Asp.

Xaa² is Arg, Lys, Ala, Cit (citrulline), Orn (ornithine), acetylatedSer, Sar, D-Arg and D-Lys. In certain embodiments, Xaa² is apositively-charged amino acid such as Arg or Lys, typically Arg.

Xaa³ is Val, Ala, Leu, Nle (norleucine), Ile, Gly, Lys, Pro, HydroxyPro(hydroxyproline), Aib (2-aminoisobutyric acid), Acpc or Tyr. In certainembodiments, Xaa³ is an aliphatic amino acid such as Val, Leu, Ile orNle, typically Val or Nle.

Xaa⁴ is Tyr, Tyr(PO₃), Thr, Ser, homoSer (homoserine), azaTyr(aza-α¹-homo-L-tyrosine) or Ala. In certain embodiments, Xaa⁴ is ahydroxyl-substituted amino acid such as Tyr, Ser or Thr, typically Tyr.

Xaa⁵ is Ile, Ala, Leu, norLeu, Val or Gly. In certain embodiments, Xaa⁵is an aliphatic amino acid such as Val, Leu, Ile or Nle, typically Ile.

Xaa⁶ is His, Arg or 6-NH₂-Phe (6-aminophenylalaine). In certainembodiments, Xaa⁶ is a fully or partially positively-charged amino acidsuch as Arg or His.

Xaa⁷ is Cys, Pro or Ala.

In certain embodiments, one or more of Xaa¹-Xaa⁷ is identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In certainsuch embodiments, all but one or two of Xaa¹-Xaa⁷ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In otherembodiments, all of Xaa¹-Xaa⁶ are identical to the corresponding aminoacid in naturally-occurring Ang-(1-7).

In certain embodiments, Xaa³ is Nle. When Xaa³ is Nle, one or more ofXaa¹-Xaa² and Xaa⁴⁻⁷ are optionally identical to the corresponding aminoacid in naturally-occurring Ang-(1-7). In certain such embodiments, allbut one or two of Xaa¹-Xaa² and Xaa⁴⁻⁷ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7). In otherembodiments, all of Xaa¹-Xaa² and Xaa⁴⁻⁷ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7), resulting inthe amino acid sequence: Asp¹-Arg²-Nle³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ IDNO:5).

In certain embodiments, the peptide has the amino acid sequenceAsp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:2) orAsp¹-Arg²-Val³-ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:6).

In some embodiments, a linear angiotensin (1-7) peptide is an Ang (1-9)peptide having a sequence ofAsp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹ (SEQ ID NO: 23). In someembodiments, an angiotensin (1-7) peptide is a derivative of Ang (1-9).For exemplary Ang (1-9) peptides, including Ang(1-9) derivatives, seeU.S. Patent Publication 2012/0172301, the disclosure of which is herebyincorporated by reference.

In some embodiments, a linear angiotensin (1-7) peptide is Alamandine,or an Alamandine derivative. Alamandine is a naturally occurring peptidewith an amino acid sequence of Ala¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQID NO: 24) that is known to be a component of the Renin-Angiotensinsystem (see Lautner et al., Discovery and Characterization ofAlamandine, 2013, Circ. Res. 112(8): 1104-1111). A discussion ofAlamandine and Alamandine derivatives may be found in European PatentApplication 2,264,048, the disclosure of which is hereby incorporated byreference.

Exemplary Cyclic Angiotensin (1-7) Peptides

In certain aspects, the invention provides a cyclic angiotensin-(1-7)(Ang-(1-7)) peptide analog comprising a linkage, such as between theside chains of amino acids corresponding to positions Tyr⁴ and Pro⁷ inAng. These peptide analogs typically comprise 7 amino acid residues, butcan also include a cleavable sequence. As discussed in greater detailbelow, the invention includes fragments and analogs where one or moreamino acids are substituted by another amino acid (including fragments).One example of such an analog is Asp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQID NO: 22), wherein a linkage is formed between Ser⁴ and Cys⁷.

Although the following section describes aspects of the invention interms of a thioether bond linking residues at the 4- and 7-positions, itshould be understood that other linkages (as described above) couldreplace the thioether bridge and that other residues could be cyclized.A thioether bridge is also referred to as a monosulfide bridge or, inthe case of Ala-S-Ala, as a lanthionine bridge. Thioetherbridge-containing peptides can be formed by two amino acids having oneof the following formulas:

In these formulae, R¹, R², R³, R⁴, R⁵ and R⁶ are independently —H, analkyl (e.g., C₁-C₆ alkyl, C₁-C₄ alkyl) or an aralkyl group, where thealkyl and aralkyl groups are optionally substituted with one or morehalogen, —OH or —NRR′ groups (where R and R′ are independently —H orC₁-C₄ alkyl). In certain embodiments, R¹, R², R³, R⁴, R⁵ and R⁶ are eachindependently —H or —CH₃, such where all are —H.

In certain embodiments, the invention provides an Ang analog orderivative comprising a thioether bridge according to formula (I).Typically, R¹, R², R³ and R⁴ are independently selected from —H and—CH₃. Peptides comprising a thioether bridge according to formula (I)can be produced, for example, by lantibiotic enzymes or by sulfurextrusion of a disulfide. In one example, the disulfide from which thesulfur is extruded can be formed by D-cysteine in position 4 andL-cysteine in position 7 or by D-cysteine in position 4 andL-penicillamine in position 7 (see, e.g., Galande, Trent and Spatola(2003) Biopolymers 71, 534-551).

In other embodiments, the linkage of the two amino acids can be thebridges depicted in Formula (II) or Formula (III). Peptides comprising athioether bridge according to Formula (II) can be made, for example, bysulfur extrusion of a disulfide formed by D-homocysteine in position 4and L-cysteine in position 7. Similarly, peptides comprising a thioetherbridge as in Formula (III) can be made, for example, by sulfur extrusionof a disulfide formed by D-cysteine in position 4 and L-homocysteine inposition 7.

As discussed above, the Ang analogs and derivatives of the inventionvary in length and amino acid composition. The Ang analogs andderivatives of the invention preferably have biological activity or arean inactive precursor molecule that can be proteolytically activated(such as how angiotensin(I), with 10 amino acids, is converted to activefragments by cleavage of 2 amino acids). The size of an Ang analog orderivative can vary but is typically between from about 5 to 10 aminoacids, as long as the “core” pentameric segment comprising the 3-7Nle-thioether-ring structure is encompassed. The amino acid sequence ofan analog or derivative of the invention can vary, typically providedthat it is biologically active or can become proteolytically activated.Biological activity of an analog or derivative can be determined usingmethods known in the art, including radioligand binding studies, invitro cell activation assays and in vivo experiments. See, for example,Godeny and Sayeski, (2006) Am. J. Physiol. Cell. Physiol.291:C1297-1307; Sarr et al., Cardiovasc. Res. (2006) 71:794-802; andKoziarz et al., (1933) Gen. Pharmacol. 24:705-713.

Ang analogs and derivatives where only the length of the peptide isvaried include the following:

a 4,7-cyclized analog designated [Cyc⁴⁻⁷]Ang-(1-7), which is derivedfrom natural Ang-(1-7) (Asp¹-Arg²-Val³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ IDNO:7).

a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-10), which isderived from natural Angiotensin I (Ang-(1-10))(Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹-Leu¹⁰, SEQ ID NO:8);

a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-8), which isderived from natural Angiotensin II (Ang-(1-8))(Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:9);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(2-8), which isderived from natural Angiotensin III (Ang-(2-8))(Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:10);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(3-8), which isderived from natural Angiotensin IV (Ang-(3-8))(Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:11);

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-7) derived fromnatural Ang-(1-7) (Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ ID NO:12);and

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷]Ang-(1-9) derived fromnatural Ang-(1-9) (Asp¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹, SEQ IDNO:13).

These analogs can have one of the thioether bridges shown in Formulae(I)-(III) as the Cyc⁴⁻⁷ moiety, for example, where Cyc⁴ and Cyc⁷ arerepresented by Formula (I), such as where R¹-R⁴ are each —H or —CH₃,typically —H.

As compared to the amino acid sequence of the natural angiotensinpeptide, the amino acids at positions 4 and 7 of the Cyc⁴⁻⁷ analog aremodified to allow introduction of the thioether-ring structures shownabove. In addition to the length of the Ang analogs, the amino acids atpositions other than 3, 4 and 7 can be the same or different from thenaturally-occurring peptide, typically provided that the analog retainsa biological function. For analogs of inactive precursors, like[Cyc⁴⁻⁷]Ang-(1-10), biological function refers to one or both of ananalog's susceptibility to angiotensin-converting enzymes that cancleave it to a biologically active fragment (e.g. Ang-(1-8) orAng-(1-7)) or the biological activity of the fragment itself. In certainembodiments, an Ang analog or derivative of the invention has nointrinsic function but inhibits the effects of one or morenaturally-occurring angiotensin compounds.

In certain embodiments, an Ang analog of the invention is represented byFormula (IV):

(IV, SEQ ID NO: 14) Xaa¹-Xaa²-Xaa³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷ 

Xaa¹ is any amino acid, but typically a negatively-charged amino acidsuch as Glu or Asp, more typically Asp.

Xaa² is a positively-charged amino acid such as Arg or Lys, typicallyArg.

Xaa³ is an aliphatic amino acid, such as Leu, Ile or Val, typically Val.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (I), (II) and(III). Typically, the R groups in Formulae (I), (II) and (III) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Ile or Val, typically Ile.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (I), (II) or (III). Cyc⁷ can be a D-stereoisomer and/or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (I), (II), (III) and (IV). Typically, the Rgroups in Formulae (I), (II),) and (III) and (IV) are —H or —CH₃,especially —H.

In certain embodiments, one or more of Xaa¹-Xaa⁶ (excluding Cyc⁴ andCyc⁷) is identical to the corresponding amino acid innaturally-occurring Ang-(1-7). In certain such embodiments, all but oneor two of Xaa¹-Xaa⁶ are identical to the corresponding amino acid innaturally-occurring Ang-(1-7). In other embodiments, all of Xaa¹-Xaa⁶are identical to the corresponding amino acid in naturally-occurringAng-(1-7).

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present in atleast one position. Thus, cyclic analogs can have a thioether linkageformed by -Ala⁴-S-Ala⁷- (Formula (I), where R¹-R⁴ are each —H);-Ala⁴-S-Abu⁷- (Formula (I): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷- (Formula (I): R¹, R³ and R⁴ are —H and R² is —CH₃).Specific examples of cyclic analogs comprise a -Abu⁴-S-Ala⁷- or-Ala⁴-S-Ala⁷- linkage.

In certain embodiments, the invention provides an Ang-(1-7) analog witha thioether-bridge between position 4 and position 7 having the aminoacid sequence Asp¹-Arg²-Val³-Abu⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:15) or theamino acid sequence Asp¹-Arg²-Val³-Ala⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:16),which are represented by the following structural diagrams:

In certain embodiments, an Ang analog or derivative of the invention isrepresented by Formula (V):

(V, SEQ ID NO: 17) Xaa¹-Xaa²-Nle³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷-Xaa⁸-Xaa⁹-Xaa¹⁰ As discussed above, one or more of Xaa¹, Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ areabsent in certain embodiments. For example, (1) Xaa¹⁰ is absent, (2)Xaa⁹ and Xaa¹⁰ are absent, (3) Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent, (4) Xaa¹is absent, (5) Xaa¹ and Xaa¹⁰ are absent, (6) Xaa¹, Xaa⁹ and Xaa¹⁰ areabsent, (7) Xaa¹, Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent, (8) Xaa¹ and Xaa² areabsent, (9) Xaa¹, Xaa² and Xaa¹⁰ are absent, (10) Xaa¹, Xaa², Xaa⁹ andXaa¹⁰ are absent, or (11) Xaa¹, Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent.For each of these embodiments, the remaining amino acids have the valuesdescribed below.

Xaa¹, when present, is any amino acid, but typically a negativelycharged amino acid such as Glu or Asp, more typically Asp.

Xaa², when present, is a positively charged amino acid such as Arg orLys, typically Arg.

Nle³ is norleucine.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (I), (II) and(III). Typically, the R groups in Formulae (I), (II) and (III) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Nle, Ile or Val, typicallyIle.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (I), (II) or (III). Cyc⁷ can be a D-stereoisomer and/or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (I), (II) and (III). Typically, the R groupsin Formulae (I), (II) and (III) are —H or —CH₃, especially —H.

Xaa⁸, when present, is an amino acid other than Pro, typically Phe orIle. In certain embodiments, Ile results in an inhibitor of Ang(1-8). Incertain embodiments, Phe maintains the biological activity of Ang(1-8)or Ang(1-10).

Xaa⁹, when present, is His.

Xaa¹⁰, when present, is an aliphatic residue, for example, Ile, Val orLeu, typically Leu.

In certain embodiments, one or more of Xaa¹-Xaa¹⁰ (excluding Nle³, Cyc⁴and Cyc⁷) is identical to the corresponding amino acid innaturally-occurring Ang (including Ang-(1-7), Ang(1-8), Ang(1-9),Ang(1-10), Ang(2-7), Ang(2-8), Ang(2-9), Ang(2-10), Ang(3-8), Ang(3-9)and Ang(3-10). In certain such embodiments, all but one or two ofXaa¹-Xaa¹⁰ (for those present) are identical to the corresponding aminoacid in naturally-occurring Ang. In other embodiments, all of Xaa¹-Xaa¹⁰(for those present) are identical to the corresponding amino acid innaturally-occurring Ang.

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present at atleast one position. Thus, encompassed are cyclic analogs comprising athioether linkage formed by -Ala⁴-S-Ala⁷-(Formula (I), where R¹-R⁴ areeach —H); -Ala⁴-S-Abu⁷- (Formula (I): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷- (Formula (I): R¹, R³ and R⁴ are —H and R² is —CH₃).Specific cyclic analogs comprise a -Abu⁴-S-Ala⁷- or -Ala⁴-S-Ala⁷-linkage.

In particular, the invention provides an Ang-(1-7) analog or derivativewith a thioether-bridge between position 4 and position 7 having theamino acid sequence Asp¹-Arg²-Nle³-Abu⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO:18) orthe amino acid sequence Asp¹-Arg²-Nle³-Ala⁴-Ile⁵-His⁶-Ala⁷ (SEQ IDNO:19).

In another aspect, the invention provides an Ang-(1-8) analog orderivative with a thioether-bridge between position 4 and position 7having Ang-(1-8) antagonistic activity, in particular an Ang(1-8) analogor derivative having the amino acid sequenceAsp¹-Arg²-Nle³-Abu⁴-Ile⁵-His⁶-Ala⁷-Ile⁸ (SEQ ID NO:20), or the aminoacid sequence Asp¹-Arg²-Nle³-Ala⁴-Ile⁵-His⁶-Ala⁷-Ile⁸ (SEQ ID NO:21).

An alkyl group is a straight chained or branched non-aromatichydrocarbon that is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10. Examples of straight chained and branched alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained orbranched alkyl group is also referred to as a “lower alkyl” group.

An aralkyl group is an alkyl group substituted by an aryl group.Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromaticgroups also include fused polycyclic aromatic ring systems in which acarbocyclic aromatic ring or heteroaryl ring is fused to one or moreother heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

Ang (1-7) Receptor Agonists

In some embodiments, the present invention provides methods of treatingbrain conditions including administering to a subject who is sufferingfrom or susceptible to one or more brain conditions an angiotensin (1-7)receptor agonist. As used herein, the term “angiotensin-(1-7) receptoragonist” encompasses any molecule that has a positive impact in afunction of an angiotensin-(1-7) receptor, in particular, the G-proteincoupled Mas receptor. In some embodiments, an angiotensin-(1-7) receptoragonist directly or indirectly enhances, strengthens, activates and/orincreases an angiotensin-(1-7) receptor (i.e., the Mas receptor)activity. In some embodiments, an angiotensin-(1-7) receptor agonistdirectly interacts with an angiotensin-(1-7) receptor (i.e., the Masreceptor). Such agonists can be peptidic or non-peptidic including,e.g., proteins, chemical compounds, small molecules, nucleic acids,antibodies, drugs, ligands, or other agents. In some embodiments, theangiotensin (1-7) receptor agonist is a non-peptidic agonist.

An exemplary class of angiotensin-(1-7) receptor agonists are1-(p-thienylbenzyl)imidazoles. Examples of these non-peptideangiotensin-(1-7) receptor agonists are represented by StructuralFormula (VI):

or pharmaceutically acceptable salts thereof, wherein:

R is halogen, hydroxyl, (C₁-C₄)-alkoxy, (C₁-C₈)-alkoxy wherein 1 to 6carbon atoms are replaced by the heteroatoms O, S, or NH (preferably byO), (C₁-C₄)-alkoxy substituted by a saturated cyclic ether such astetrahydropyran or tetrahydrofuran, O—(C₁-C₄)-alkenyl,O—(C₁-C₄)-alkylaryl, or aryloxy that is unsubstituted or substituted bya substituent selected from halogen, (C₁-C₃)-alkyl, (C₁-C₃)-alkoxy andtrifluoromethyl;

R² is CHO, COOH, or (3) CO—O—(C₁-C₄)-alkyl;

R³ is (C₁-C₄)-alkyl or aryl;

R⁴ is hydrogen, halogen (chloro, bromo, fluoro), or (C₁-C₄)-alkyl;

X is oxygen or sulfur;

Y is oxygen or —NH—;

R⁵ is hydrogen, (C₁-C₆)-alkyl; or (C₁-C₄)-alkylaryl, where R⁵ ishydrogen when Y is —NH—; and

R⁶ is (C₁-C₅)-alkyl.

In certain embodiments, R¹ is not halogen when R² is COOH orCO—O—(C₁-C₄)-alkyl.

In some embodiments, an angiotensin-(1-7) receptor agonist is AVE 0991,5-formyl-4-methoxy-2-phenyl-1[[4-[2-(ethylaminocarbonylsulfonamido)-5-isobutyl-3-thienyl]-phenyl]-methyl]-imidazole,which is represented by the following structure:

Another exemplary class of angiotensin-(1-7) receptor agonists arep-thienylbenzylamides. Examples of these non-peptide angiotensin-(1-7)receptor agonists are represented by Structural Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is (C₁-C₅)-alkyl that is unsubstituted or substituted by a radicalchosen from NH₂, halogen, O—(C₁-C₃)-alkyl, CO—O—(C₁-C₃)-alkyl and CO₂H,(C₃-C₈)-cycloalkyl, (C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, (C₆-C₁₀)-aryl thatis unsubstituted or substituted by a radical chosen from halogen andO—(C₁-C₃)-alkyl, (C₁-C₃)-alkyl-(C₆-C₁₀)-aryl where the aryl radical isunsubstituted or substituted by a radical chosen from halogen andO—(C₁-C₃)-alkyl, (C₁-C₅)-heteroaryl, or(C₁-C₃)-alkyl-(C₁-C₅)-heteroaryl;

R² is hydrogen, (C₁-C₆)-alkyl that is unsubstituted or substituted by aradical chosen from halogen and O—(C₁-C₃)-alkyl, (C₃-C₈)-cycloalkyl,(C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, (C₆-C₁₀)-aryl that is unsubstituted orsubstituted by a radical chosen from among halogen, 0-(C₁-C₃)-alkyl andCO—O—(C₁-C₃)-alkyl, or (C₁-C₃)-alkyl-(C₆-C₁₀)-aryl that is unsubstitutedor substituted by a radical chosen from halogen and O—(C₁-C₃)-alkyl;

R³ is hydrogen, COOH, or COO—(C₁-C₄)-alkyl;

R⁴ is hydrogen, halogen; or (C₁-C₄)-alkyl;

R⁵ is hydrogen or (C₁-C₆)-alkyl;

R⁶ is hydrogen, (C₁-C₆)-alkyl, (C₁-C₃)-alkyl-(C₃-C₈)-cycloalkyl, or(C₂-C₆)-alkenyl; and

X is oxygen or NH.

Additional examples of angiotensin-(1-7) receptor agonists are describedin U.S. Pat. No. 6,235,766, the contents of which are incorporated byreference herein.

Various angiotensin-(1-7) receptor agonists described above can bepresent as pharmaceutically acceptable salts. As used herein, “apharmaceutically acceptable salt” refers to salts that retain thedesired activity of the peptide or equivalent compound, but preferablydo not detrimentally affect the activity of the peptide or othercomponent of a system, which uses the peptide. Examples of such saltsare acid addition salts formed with inorganic acids, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid,nitric acid, and the like. Salts may also be formed with organic acidssuch as, for example, acetic acid, oxalic acid, tartaric acid, succinicacid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid,ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,polyglutamic acid, and the like. Salts formed from a cationic materialmay utilize the conjugate base of these inorganic and organic acids.Salts may also be formed with polyvalent metal cations such as zinc,calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickeland the like or with an organic cation formed fromN,N′-dibenzylethylenediamine or ethylenediamine, or combinations thereof(e.g., a zinc tannate salt). The non-toxic, physiologically acceptablesalts are preferred.

The salts can be formed by conventional means such as by reacting thefree acid or free base forms of the product with one or more equivalentsof the appropriate acid or base in a solvent or medium in which the saltis insoluble, or in a solvent such as water which is then removed invacuo or by freeze-drying, or by exchanging the cations of an existingsalt for another cation on a suitable ion exchange resin.

An alkyl group is a straight chained or branched non-aromatichydrocarbon that is completely saturated. Typically, a straight chainedor branched alkyl group has from 1 to about 20 carbon atoms, preferablyfrom 1 to about 10. Examples of straight chained and branched alkylgroups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl,tert-butyl, pentyl, hexyl, pentyl and octyl. A C1-C4 straight chained orbranched alkyl group is also referred to as a “lower alkyl” group.

An alkenyl group is a straight chained or branched non-aromatichydrocarbon that is includes one or more double bonds. Typically, astraight chained or branched alkenyl group has from 2 to about 20 carbonatoms, preferably from 2 to about 10. Examples of straight chained andbranched alkenyl groups include ethenyl, n-propenyl, and n-butenyl.

Aromatic (aryl) groups include carbocyclic aromatic groups such asphenyl, naphthyl, and anthracyl, and heteroaryl groups such asimidazolyl, thienyl, furyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl,pyrrolyl, pyrazinyl, thiazolyl, oxazolyl, and tetrazolyl. Aromaticgroups also include fused polycyclic aromatic ring systems in which acarbocyclic aromatic ring or heteroaryl ring is fused to one or moreother heteroaryl rings. Examples include benzothienyl, benzofuryl,indolyl, quinolinyl, benzothiazole, benzoxazole, benzimidazole,quinolinyl, isoquinolinyl and isoindolyl.

An aralkyl group is an alkyl group substituted by an aryl group.

Formulations

In accordance with the methods of the invention, an Ang (1-7) peptide orangiotensin (1-7) receptor agonist as described herein of the inventioncan be administered to a subject alone (e.g., as a purified peptide orcompound), or as a component of a composition or medicament (e.g., inthe manufacture of a medicament for the treatment of the disease), asdescribed herein. The compositions can be formulated with aphysiologically acceptable carrier or excipient to prepare apharmaceutical composition. The carrier and composition can be sterile.The formulation should suit the mode of administration, for exampleintravenous or subcutaneous administration. Methods of formulatingcompositions are known in the art (see, e.g., Remington'sPharmaceuticals Sciences, 17^(th) Edition, Mack Publishing Co., (AlfonsoR. Gennaro, editor) (1989)).

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring and/oraromatic substances and the like) which do not deleteriously react withthe active compounds or interference with their activity. In a preferredembodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, sustainedrelease formulation, or powder. The composition can also be formulatedas a suppository, with traditional binders and carriers such astriglycerides.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

In some embodiments, provided compositions, including those provided aspharmaceutical formulations, comprise a liquid carrier such as but notlimited to water, saline, phosphate buffered saline, Ringer's solution,dextrose solution, serum-containing solutions, Hank's solution, otheraqueous physiologically balanced solutions, oils, esters and glycols.

An Ang (1-7) peptide or angiotensin (1-7) receptor agonist as describedherein can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

Oral Formulations

In some embodiments, a suitable pharmaceutical composition is an oralformulation. It is contemplated that any medically-acceptable oralformulation may be used within the scope of the present invention.

In some embodiments, provided compositions include at least onepH-lowering agent. It is contemplated that a pH-lowering agent suitablefor use in some embodiments of the present invention include anypharmaceutically acceptable pH-lowering agent, or combination ofpH-lowering agents, that are a) not toxic to the gastrointestinal tract,b) are capable of either delivering hydrogen ions or capable of inducinghigher hydrogen ion content from the local environment, and/or c) thatare capable of being orally administered in an amount sufficient tolower the local intestinal pH below the pH optima for proteases foundthere. Various tests may be used to determine if a pH-lowering agent issuitable for the present invention and what amount is appropriate. Forexample, a pH-lowering agent or combination of pH-lowering agents issuitable for the present invention if a particular amount, when added toa solution of 10 milliliters of 0.1M sodium bicarbonate lowers the pH ofthe solution to no higher than 5.5, 4.7, or 3.5. In some embodiments, anamount of pH-lowering agent or agents may be added to lower pH, in asolution of 10 milliliters of 0.1M sodium bicarbonate, to no higher than3.4, 3.2, 3.0, or 2.8.

In some embodiments, a suitable pH-lowering agent or agents include atleast one pH-lowering agent that has a pKa no higher than 4.2 (e.g., nohigher than 4.0, 3.8, 3.6, 3.4, 3.2, 3.0 or 2.8). Exemplary pH-loweringagents suitable for the present invention include, but are not limitedto, carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric,fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic,isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic,propionic, pyruvic, succinic, tartaric, and valeric; aluminum chloride;zinc chloride; acid salts of amino acids (or derivatives thereof)including acid salts of acetylglutamic acid, alanine, arginine,asparagine, aspartic acid, betaine, carnitine, carnosine, citrulline,creatine, glutamic acid, glycine, histidine, hydroxylysine,hydroxyproline, hypotaurine, isoleucine, leucine, lysine,methylhistidine, norleucine, ornithine, phenylalanine, proline,sarcosine, serine, taurine, threonine, tryptophan, tyrosine, and valine;certain phosphate esters including fructose 1,6 diphosphate and glucose1,6 diphosphate may also be appropriate pH-lowering agents in certainembodiments. In particular embodiments, citric acid or tartaric acid isused as pH-lowering agent.

The quantity required of any particular pH-lowering agent or combinationof pH-lowering agents may vary. Typically, a suitable amount may bedetermined using various tests known in the art and described herein(for example, using pH-lowering test in a solution of 10 milliliters of0.1M sodium bicarbonate described above). As non-limiting examples,suitable amount of a pH lowering agent used in a formulation accordingto the present invention may be an amount of or greater than about 100mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675, mg, 700 mg, 725mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950mg, 975 mg, or 1,000 mg. In other embodiments, the amount of citric acidused may exceed 1,000 mg.

In some embodiments, a suitable amount of a pH lowering agent (e.g.,citric acid or tartaric acid) used may be measured as a percent of thetotal weight of a particular dosage form. As non-limiting examples, asuitable amount of a pH lowering agent used may be an amount of orgreater than about 10% (e.g., of or greater than 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) ofthe total weight of a solid dosage form.

In various embodiments, a composition of the invention includes one ormore absorption enhancers. As used herein, an absorption enhancer refersto an agent that increase the solubility of other components in eitherthe aqueous or lipophilic environment into which they are releasedand/or enhance the uptake of an active peptide (e.g., an angiotensin(1-7) peptide) across the intestinal wall. In some embodiments, anabsorption enhancer is referred to as a solubility enhancer and/or anuptake enhancer.

In some embodiments, it is possible to have a mixture of absorptionenhancers wherein some provide enhanced solubility, some provideenhanced uptake, and some provide both. It is possible to have variousnumbers of absorption enhancers in a given embodiment including, withoutlimitation, one, two, three, four, five, six, seven, eight, nine, or tenabsorption enhancers.

Surface active agents are an example of useful absorption enhancers withproperties of both solubility enhancers and uptake enhancers. In someembodiment, when surface active agents are used as absorption enhancers,they may be free flowing powders for facilitating the mixing and loadingof capsules during the manufacturing process. In other embodiments whena surface active agent is used to increase the bioavailability of anangiotensin (1-7) peptide, the surface active agent may be selected fromthe group consisting of (a) anionic surface active agents such ascholesterol derivatives (e.g. bile acids), (b) cationic surface agents(e.g. acyl carnitines, phospholipids and the like), (c) non-ionicsurface active agents, and (d) mixtures of anionic surface active agentsand negative charge neutralizers, and combinations thereof. Negativecharge neutralizers include but are not limited to acyl carnitines,cetyl pyridium chloride, and the like.

In some embodiments, an acid soluble bile acid and a cationic surfaceactive agent with be used together as absorption enhancers. Acylcarnitines (such as lauroyl carnitine), phospholipids and bile acids maybe particularly effective absorption enhancers in some embodiments.

While a variety of absorption enhancers are suitable for use in variousembodiments, the following exemplary list is intended to illustrate someembodiments of the present invention. Without limitation, some suitableabsorption enhancers include: (a) salicylates such as sodium salicylate,3-methoxysalicylate, 5-methoxysalicylate and homovanilate; (b) bileacids such as taurocholic, tauorodeoxycholic, deoxycholic, cholic,glycholic, lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic,dehydrocholic, fusidic, etc.; (c) non-ionic surfactants such aspolyoxyethylene ethers (e.g. Brij 36T, Brij 52, Brij 56, Brij 76, Brij96, Texaphor A6, Texaphor A14, Texaphor A60 etc.), p-t-octyl phenolpolyoxyethylenes (Triton X-45, Triton X-100, Triton X-114, Triton X-305etc.) nonylphenoxypoloxyethylenes (e.g. Igepal CO series),polyoxyethylene sorbitan esters (e.g. Tween-20, Tween-80 etc.); (d)anionic surfactants such as dioctyl sodium sulfosuccinate; (e)lyso-phospholipids such as lysolecithin andlysophosphatidylethanolamine; (f) acylcarnitines, acylcholines and acylamino acids such as lauroylcarnitine, myristoylcarnitine,palmitoylcarnitine, lauroylcholine, myristoylcholine, palmitoylcholine,hexadecyllysine, N-acylphenylalanine, N-acylglycine etc.; g) watersoluble phospholipids such as diheptanoylphosphatidylcholine,dioctylphosphatidylcholine etc.; (h) medium-chain glycerides which aremixtures of mono-, di- and triglycerides containing medium-chain-lengthfatty acids (caprylic, capric and lauric acids); (i)ethylene-diaminetetraacetic acid; (j) cationic surfactants such ascetylpyridinium chloride; (k) fatty acid derivatives of polyethyleneglycol such as Labrasol, Labrafac, etc.; and (l) alkylsaccharides suchas lauroyl maltoside, lauroyl sucrose, myristoyl sucrose, palmitoylsucrose, etc.

In some embodiments, the absorption enhancer(s) will be present in aquantity measured as a percent by weight, relative to the overall weightof the pharmaceutical composition (typically exclusive of entericcoating). By way of additional non-limiting example, the quantity ofabsorption enhancer present in an embodiment may range from 0.1 to 20percent by weight; from 0.5 to 20 percent by weight; from 1.0 to 20percent by weight, from 2.0 to 20 percent by weight, from 3.0 to 20percent by weight, from 4.0 to 20 percent by weight, from from 5.0 to 20percent by weight, from 5.0 to 15 percent by weight, from 5.0 to 14percent by weight, from 5.0 to 13 percent by weight, from 5.0 to 12percent by weight, from 5.0 to 12 percent by weight, from 5.0 to 11percent by weight, from 5.0 to 10 percent by weight, from 6.0 to 10percent by weight, from 7.0 to 10 percent by weight, from 8.0 to 10percent by weight, from 9.0 to 10 percent by weight, from 5.0 to 9.0percent by weight, from 5.0 to 8.0 percent by weight, from 5.0 to 7.0percent by weight, and from 5.0 to 6.0 percent by weight.

In some embodiments, the weight ratio of pH-lowering agent(s) toabsorption enhancer(s) may be about 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 orbetween any two of the foregoing exemplary ratios. The total weight ofall pH-lowering agents and the total weight of all absorption enhancersin a given pharmaceutical composition is included in the foregoingexemplary ratios. For example, if a pharmaceutical composition includestwo pH-lowering agents and three absorption enhancers, the foregoingratios will be computed on the total combined weight of both pH-loweringagents and the total combined weight of all three absorption enhancers.

In some embodiments, the absorption enhancer(s) will be soluble at acidpH, such as less than pH 5.5, and in particular, between pH 3.0 and pH5.0.

In some embodiments, provided compositions comprise one or moreprotective vehicles. As used herein, a protective vehicle refers to anyprotective component and/or structure, such as a carrier, a layer, acoating or other vehicle, that protects an active peptide (e.g., anangiotensin (1-7) peptide) from stomach proteases. Typically, aprotective vehicle dissolves eventually so that the active and otheringredients in a particular dosage form may be released. A common formof protective vehicle is an enteric coating. In some embodiments, asuitable enteric costing may prevent breakdown of the pharmaceuticalcomposition of the invention in 0.1N HCl for at least two hours, thencapable of permitting complete release of all contents of thepharmaceutical composition within thirty minutes after pH is increasedto 6.3 in a dissolution bath in which said composition is rotating at100 revolutions per minute.

Many enteric coatings are known in the art and are useful in one or moreembodiments. Non-limiting examples of enteric coatings include celluloseacetate phthalate, hydroxypropyl methylethylcellulose succinate,hydroxypropyl methylcellulose phthalate, carboxyl methylethylcelluloseand methacrylic acid-methyl methacrylate copolymer. In some embodiments,an angiotensin (1-7) peptide, absorption enhancers such as solubilityand/or uptake enhancer(s), and pH-lowering agent(s), are included in asufficiently viscous protective syrup to permit protected passage of thecomponents of the embodiment through the stomach.

Suitable enteric coatings may be applied, for example, to capsules afterthe active and other components of the invention have been loaded withinthe capsule. In other embodiments, enteric coating is coated on theoutside of a tablet or coated on the outer surface of particles ofactive components which are then pressed into tablet form, or loadedinto a capsule.

In some embodiments it may be desirable that all components of theinvention be released from the carrier or vehicle, and solubilized inthe intestinal environment as simultaneously as possible. It may also bepreferred in some embodiments that the vehicle or carrier release theactive components in the small intestine where uptake enhancers thatincrease transcellular or paracellular transport are less likely tocause undesirable side effects than if the same uptake enhancers werelater released in the colon. It will be appreciated, however, that thepresent invention is believed effective in the colon as well as in thesmall intestine. Numerous vehicles or carriers, in addition to the onesdiscussed above, are known in the art.

In some embodiments, it may be desirable (especially in optimizing howsimultaneously the components of the invention are released) to keep theamount of enteric coating low. In some embodiments, an enteric coatingadds no more than 30% to the weight of the remainder of pharmaceuticalcomposition such as a solid dosage form (the “remainder” being thepharmaceutical composition exclusive of enteric coating itself). Inother embodiments, an enteric coating adds less than 20%, less than 19%,less than 18%, less than 17%, less than 16%, less than 15%, less than14%, less than 13%, less than 12%, less than 11%, or less than 10%. Insome embodiments, a protective vehicle such as an enteric coatingconstitutes an amount of or less than approximately 25%, 24%, 23%, 22%,21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5% of the total weight of a pharmaceutical composition (e.g., asolid dosage form).

In some embodiments, a composition is administered in a therapeuticallyeffective amount and/or according to a dosing regimen that is correlatedwith a particular desired outcome (e.g., with treating or reducing riskfor Marfan Syndrome or a Marfan-related disorder).

Particular doses or amounts to be administered in accordance with thepresent invention may vary, for example, depending on the nature and/orextent of the desired outcome, on particulars of route and/or timing ofadministration, and/or on one or more characteristics (e.g., weight,age, personal history, genetic characteristic, lifestyle parameter,severity of cardiac defect and/or level of risk of cardiac defect, etc.,or combinations thereof). Such doses or amounts can be determined bythose of ordinary skill. In some embodiments, an appropriate dose oramount is determined in accordance with standard clinical techniques.For example, in some embodiments, an appropriate dose or amount is adose or amount sufficient to reduce a disease severity index score by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more.For example, in some embodiments, an appropriate dose or amount is adose or amount sufficient to reduce a disease severity index score by 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%.Alternatively or additionally, in some embodiments, an appropriate doseor amount is determined through use of one or more in vitro or in vivoassays to help identify desirable or optimal dosage ranges or amounts tobe administered.

In various embodiments, an Ang (1-7) peptide or angiotensin (1-7)receptor agonist is administered at a therapeutically effective amount.As used herein, the term “therapeutically effective amount” is largelydetermined based on the total amount of the therapeutic agent containedin the pharmaceutical compositions of the present invention. Generally,a therapeutically effective amount is sufficient to achieve a meaningfulbenefit to the subject (e.g., treating, modulating, curing, preventingand/or ameliorating the underlying disease or condition). In someparticular embodiments, appropriate doses or amounts to be administeredmay be extrapolated from dose-response curves derived from in vitro oranimal model test systems.

Therapeutically effective dosage amounts of angiotensin (1-7) peptidesor angiotensin (1-7) receptor agonists, including derivatives, analogs,and/or salts may be present in varying amounts in various embodiments.For example, in some embodiments, a therapeutically effective amount ofan angiotensin (1-7) peptide may be an amount ranging from about 10-1000mg (e.g., about 20 mg-1,000 mg, 30 mg-1,000 mg, 40 mg-1,000 mg, 50mg-1,000 mg, 60 mg-1,000 mg, 70 mg-1,000 mg, 80 mg-1,000 mg, 90 mg-1,000mg, about 10-900 mg, 10-800 mg, 10-700 mg, 10-600 mg, 10-500 mg,100-1000 mg, 100-900 mg, 100-800 mg, 100-700 mg, 100-600 mg, 100-500 mg,100-400 mg, 100-300 mg, 200-1000 mg, 200-900 mg, 200-800 mg, 200-700 mg,200-600 mg, 200-500 mg, 200-400 mg, 300-1000 mg, 300-900 mg, 300-800 mg,300-700 mg, 300-600 mg, 300-500 mg, 400 mg-1,000 mg, 500 mg-1,000 mg,100 mg-900 mg, 200 mg-800 mg, 300 mg-700 mg, 400 mg-700 mg, and 500mg-600 mg). In some embodiments, an angiotensin (1-7) peptide orangiotensin (1-7) receptor agonist is present in an amount of or greaterthan about 10 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg,400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg.In some embodiments, an angiotensin (1-7) peptide or angiotensin (1-7)receptor agonist is present in an amount of or less than about 1000 mg,950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg,500 mg, 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, or 100mg. In some embodiments, the therapeutically effective amount describedherein is provided in one dose. In some embodiments, the therapeuticallyeffective amount described herein is provided in one day.

In other embodiments, a therapeutically effective dosage amount may be,for example, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., fromabout 0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kgweight to 300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kgweight, from about 0.001 mg/kg weight to 100 mg/kg weight, from about0.001 mg/kg weight to 90 mg/kg weight, from about 0.001 mg/kg weight to80 mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, fromabout 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kgweight to 50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kgweight, from about 0.001 mg/kg weight to 30 mg/kg weight, from about0.001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to20 mg/kg weight, from about 0.001 mg/kg weight to 15 mg/kg weight, fromabout 0.001 mg/kg weight to 10 mg/kg weight. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.001 mg/kg weight to about 1 mg/kg weight,e.g. from about 0.001 mg/kg weight to about 0.9 mg/kg weight, from about0.001 mg/kg weight to about 0.8 mg/kg weight, from about 0.001 mg/kgweight to about 0.8 mg/kg weight, from about 0.001 mg/kg weight to about0.7 mg/kg weight, from about 0.001 mg/kg weight to about 0.6 mg/kgweight, from about 0.001 mg/kg weight to about 0.5 mg/kg weight, fromabout 0.01 mg/kg weight to about 1 mg/kg weight, from about 0.01 mg/kgweight to about 0.9 mg/kg weight, from about 0.01 mg/kg weight to about0.8 mg/kg weight, from about 0.01 mg/kg weight to about 0.7 mg/kgweight, from about 0.01 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.01 mg/kg weight to about 0.5 mg/kg weight, from about 0.02 mg/kgweight to about 1 mg/kg weight, from about 0.02 mg/kg weight to about0.9 mg/kg weight, from about 0.02 mg/kg weight to about 0.8 mg/kgweight, from about 0.02 mg/kg weight to about 0.7 mg/kg weight, fromabout 0.02 mg/kg weight to about 0.6 mg/kg weight, from about 0.02 mg/kgweight to about 0.5 mg/kg weight, from about 0.03 mg/kg weight to about1 mg/kg weight, from about 0.03 mg/kg weight to about 0.9 mg/kg weight,from about 0.03 mg/kg weight to about 0.8 mg/kg weight, from about 0.03mg/kg weight to about 0.7 mg/kg weight, from about 0.03 mg/kg weight toabout 0.6 mg/kg weight, from about 0.03 mg/kg weight to about 0.5 mg/kgweight, from about 0.04 mg/kg weight to about 1 mg/kg weight, from about0.04 mg/kg weight to about 0.9 mg/kg weight, from about 0.04 mg/kgweight to about 0.8 mg/kg weight, from about 0.04 mg/kg weight to about0.7 mg/kg weight, from about 0.04 mg/kg weight to about 0.6 mg/kgweight, from about 0.04 mg/kg weight to about 0.5 mg/kg weight, fromabout 0.05 mg/kg weight to about 1 mg/kg weight, from about 0.05 mg/kgweight to about 0.9 mg/kg weight, from about 0.05 mg/kg weight to about0.8 mg/kg weight, from about 0.05 mg/kg weight to about 0.7 mg/kgweight, from about 0.05 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.05 mg/kg weight to about 0.5 mg/kg weight. In some embodiments,the therapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g.from about 0.0001 mg/kg weight to 0.09 mg/kg weight, from about 0.0001mg/kg weight to 0.08 mg/kg weight, from about 0.0001 mg/kg weight to0.07 mg/kg weight, from about 0.0001 mg/kg weight to 0.06 mg/kg weight,from about 0.0001 mg/kg weight to 0.05 mg/kg weight, from about 0.0001mg/kg weight to about 0.04 mg/kg weight, from about 0.0001 mg/kg weightto 0.03 mg/kg weight, from about 0.0001 mg/kg weight to 0.02 mg/kgweight, from about 0.0001 mg/kg weight to 0.019 mg/kg weight, from about0.0001 mg/kg weight to 0.018 mg/kg weight, from about 0.0001 mg/kgweight to 0.017 mg/kg weight, from about 0.0001 mg/kg weight to 0.016mg/kg weight, from about 0.0001 mg/kg weight to 0.015 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.014 mg/kg weight, from about 0.0001 mg/kgweight to 0.013 mg/kg weight, from about 0.0001 mg/kg weight to 0.012mg/kg weight, from about 0.0001 mg/kg weight to 0.011 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.01 mg/kg weight, from about 0.0001 mg/kgweight to 0.009 mg/kg weight, from about 0.0001 mg/kg weight to 0.008mg/kg weight, from about 0.0001 mg/kg weight to 0.007 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.006 mg/kg weight, from about 0.0001 mg/kgweight to 0.005 mg/kg weight, from about 0.0001 mg/kg weight to 0.004mg/kg weight, from about 0.0001 mg/kg weight to 0.003 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.002 mg/kg weight. In some embodiments,the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002mg/kg weight, 0.0003 mg/kg weight, 0.0004 mg/kg weight, 0.0005 mg/kgweight, 0.0006 mg/kg weight, 0.0007 mg/kg weight, 0.0008 mg/kg weight,0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kgweight, 0.004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight,0.007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kgweight, 0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05mg/kg weight, 0.06 mg/kg weight, 0.07 mg/kg weight, 0.08 mg/kg weight,0.09 mg/kg weight, or 0.1 mg/kg weight. The effective dose for aparticular individual can be varied (e.g., increased or decreased) overtime, depending on the needs of the individual.

In some embodiments, the angiotensin (1-7) peptide is administered at aneffective dose ranging from about 1-1,000 μg/kg/day (e.g., ranging fromabout 1-900 μg/kg/day, 1-800 μg/kg/day, 1-700 μg/kg/day, 1-600μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg/day, 1-40 μg/kg/day, 1-30μg/kg/day, 1-20 μg/kg/day, 1-10 μg/kg/day). In some embodiments, theangiotensin (1-7) peptide is administered at an effective dose rangingfrom about 1-500 μg/kg/day. In some embodiments, the angiotensin (1-7)peptide is administered at an effective dose ranging from about 1-100μg/kg/day. In some embodiments, the angiotensin (1-7) peptide isadministered at an effective dose ranging from about 1-60 μg/kg/day. Insome embodiments, the angiotensin (1-7) peptide is administered at aneffective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, or 1,000 μg/kg/day.

Routes of Administration

An angiotensin (1-7) peptide or angiotensin (1-7) receptor agonist asdescribed herein (or a composition or medicament containing anangiotensin (1-7) peptide or Angiotensin (1-7) receptor agonist asdescribed herein) may be administered by any appropriate route. In someembodiments, the angiotensin (1-7) peptide is administered parenterally.In some embodiments, the parenteral administration is selected fromintravenous, intradermal, inhalation, transdermal (topical),intraocular, intramuscular, subcutaneous, intramuscular, and/ortransmucosal administration. In some embodiments, an angiotensin (1-7)peptide or angiotensin (1-7) receptor agonist as described herein isadministered subcutaneously. As used herein, the term “subcutaneoustissue”, is defined as a layer of loose, irregular connective tissueimmediately beneath the skin. For example, the subcutaneousadministration may be performed by injecting a composition into areasincluding, but not limited to, thigh region, abdominal region, glutealregion, or scapular region. In some embodiments, an angiotensin (1-7)peptide or angiotensin (1-7) receptor agonist as described herein isadministered intravenously. In other embodiments, an angiotensin (1-7)peptide or angiotensin (1-7) receptor agonist as described herein isadministered by direct administration to a target tissue, such as heartor muscle (e.g., intramuscular), tumor (intratumorally), nervous system(e.g., direct injection into the brain; intraventricularly;intrathecally). Alternatively, an angiotensin (1-7) peptide orangiotensin (1-7) receptor agonist as described herein (or a compositionor medicament containing an angiotensin (1-7) peptide or angiotensin(1-7) receptor agonist as described herein) can be administered byinhalation, parenterally, intradermally, transdermally, ortransmucosally (e.g., orally or nasally). More than one route can beused concurrently, if desired.

In some embodiments, an angiotensin (1-7) peptide or angiotensin (1-7)receptor agonist as described herein is administered orally. In someembodiments, the present invention provides solid dosage forms of anangiotensin (1-7) peptide or angiotensin (1-7) receptor agonist asdescribed herein for oral administration including (a) an angiotensin(1-7) peptide, (b) at least one pharmaceutically acceptable pH-loweringagent, (c) at least one absorption enhancer effective to promotebioavailability of the angiotensin (1-7) peptide, and (d) a protectivevehicle. In some embodiments, the solid dosage form is a capsule ortablet. Various methods and ingredients for making oral formulations areknown in the art and it is expected that one of skill would be able todetermine which of these methods and ingredients will be compatible withthe invention as described in this specification and/or in U.S.Provisional Patent Application Ser. No. 61/701,972, filed on Sep. 17,2012, the disclosure of which is hereby incorporated in its entirety.Such methods and ingredients are also contemplated as within the scopeof the present invention.

Dosing Schedules

Various embodiments may include differing dosing regimen. In someembodiments, the angiotensin (1-7) peptide or angiotensin (1-7) receptoragonist is administered via continuous infusion. In some embodiments,the continuous infusion is intravenous. In other embodiments, thecontinuous infusion is subcutaneous. Alternatively or additionally, insome embodiments, the angiotensin (1-7) peptide or angiotensin (1-7)receptor agonist is administered bimonthly, monthly, twice monthly,triweekly, biweekly, weekly, twice weekly, thrice weekly, daily, twicedaily, or on another clinically desirable dosing schedule. The dosingregimen for a single subject need not be at a fixed interval, but can bevaried over time, depending on the needs of the subject.

Combination Therapies

In some embodiments, an Ang (1-7) peptide or angiotensin (1-7) receptoragonist will be used as a part of a combination therapy. It iscontemplated that any known therapeutic or treatment for one or morebrain conditions may be used with one or more Ang (1-7) peptides orangiotensin (1-7) receptor agonists as disclosed herein. Exemplarycompounds that may be used with one or more Ang (1-7) peptides orangiotensin (1-7) receptor agonists as a combination therapy include,but are not limited to, beta blockers, calcium channel blockers, ACEinhibitors, angiotensin II receptor antagonists (e.g. losartan),anticoagulants, and combinations thereof.

Kits

In some embodiments, the present invention further provides kits orother articles of manufacture which contains an Ang (1-7) peptide, anangiotensin (1-7) receptor agonist or a formulation containing the sameand provides instructions for its reconstitution (if lyophilized) and/oruse. Kits or other articles of manufacture may include a container, asyringe, vial and any other articles, devices or equipment useful inadministration (e.g., subcutaneous, by inhalation). Suitable containersinclude, for example, bottles, vials, syringes (e.g., pre-filledsyringes), ampules, cartridges, reservoirs, or lyo-jects. The containermay be formed from a variety of materials such as glass or plastic. Insome embodiments, a container is a pre-filled syringe. Suitablepre-filled syringes include, but are not limited to, borosilicate glasssyringes with baked silicone coating, borosilicate glass syringes withsprayed silicone, or plastic resin syringes without silicone.

Typically, the container may holds formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor, for example, subcutaneous administration. In some embodiments, acontainer may contain a single dose of a stable formulation containingan Ang (1-7) peptide or angiotensin (1-7) receptor agonist. In variousembodiments, a single dose of the stable formulation is present in avolume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml,2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml. Alternatively, a containerholding the formulation may be a multi-use vial, which allows for repeatadministrations (e.g., from 2-6 administrations) of the formulation.Kits or other articles of manufacture may further include a secondcontainer comprising a suitable diluent (e.g., BWFI, saline, bufferedsaline). Upon mixing of the diluent and the formulation, the finalprotein concentration in the reconstituted formulation will generally beat least 1 mg/ml (e.g., at least 5 mg/ml, at least 10 mg/ml, at least 20mg/ml, at least 30 mg/ml, at least 40 mg/ml, at least 50 mg/ml, at least75 mg/ml, at least 100 mg/ml). Kits or other articles of manufacture mayfurther include other materials desirable from a commercial and userstandpoint, including other buffers, diluents, filters, needles,syringes, and package inserts with instructions for use. In someembodiments, kits or other articles of manufacture may include aninstruction for self-administration.

EXAMPLES Example 1—Angiotensin (1-7) Peptides in Decreasing Aortic RingDilation

In this Example, FBN1^(C1039G/+) mice, a known and accepted model ofMarfan Syndrome, are used to assess the effects of several angiotensin(1-7) peptides and an angiotensin (1-7) receptor agonist, AVE0991, onthe aortic dilation typically seen in these mice. See, Xiong et al.,Doxycycline delays aneurysm rupture in a mouse model of Marfan Syndrome,2008, J. Vascular Surg 47:166-172. This mutation of the fibrillin-1(FBN1) gene leads these mice to develop progressive aortic root dilationwith sporadic aortic dissections throughout life, often leading todeath.

In this example, three angiotensin (1-7) peptides, namely, TXA127 (SEQID NO:1), PanCyte (SEQ ID NO:22), and TXA301 (SEQ ID NO: 2) and a smallmolecule angiotensin (1-7) receptor agonist AVE0991 are used to examinetheir effect on the progression of aortic dilation over time.Specifically, 110 FBN1^(C1039G/+) mice, (10 per group) are placed intoone of the groups outlined in Table 1 below.

TABLE 1 Study Design Route of Dosing Group Agent Dose N Admin. Frequency1 Vehicle PBS 10 Subcutaneous Daily Control (PBS) 2 TXA127 50 10Subcutaneous Daily μg/kg/day 3 TXA127 500 10 Subcutaneous Dailyμg/kg/day 4 TXA127 1,000 10 Subcutaneous Daily μg/kg/day 5 PanCyte 50 10Subcutaneous Daily μg/kg/day 6 PanCyte 500 10 Subcutaneous Dailyμg/kg/day 7 PanCyte 1,000 10 Subcutaneous Daily μg/kg/day 8 TXA301 50 10Subcutaneous Daily μg/kg/day 9 TXA301 500 10 Subcutaneous Dailyμg/kg/day 10 TXA301 1,000 10 Subcutaneous Daily μg/kg/day 11 AVE0991 30010 Subcutaneous Daily μg/kg/day

In this example, TXA127, TXA301, PanCyte, and AVE0991 are prepared insaline and administrated subcutaneously via subscapular injection at adose volume of 100 μl/mouse daily for twenty-eight days. Dosingsolutions are prepared fresh every three days.

One week after the final injection, each animal is sacrificed, and theaortic ring is removed and analyzed to determine if administration of anangiotensin (1-7) peptide or angiotensin (1-7) receptor agonist issufficient to decrease the degree of aortic dilation of treated mice ascompared to control animals.

It is expected that administration of an angiotensin (1-7) peptidedecreases aortic ring dilation in FBN1^(C1039G/+) mice. Administrationof an angiotensin (1-7) peptide will also decrease the degree of jointlaxity in FBN1^(C1039G/+) mice.

Example 2—TXA127 in Decreasing Aortic Aneurysm Growth

In this Example, the ability of TXA127 to attenuate aortic root growthtypically observed in Marfan sufferers is described. Aortic root growthis one of the most serious, and often deadly, effects of the disease.

In this Example, a total of 20 mice were used, with 8 mice beingwild-type and 12 being Fbn1^(C1039G/+) mice, also referred to as “Marfanmice”, that are each homozygous for a fibrillin-1 (Fbn1) allele encodinga cysteine substitution, Cys1039Gly (C1039G), in an epidermal growthfactor-like domain of Fbn1 (Fbn1^(C1039G/+).

The Fbn1C1039G mice are a well-accepted model of Marfan Syndrome and theFbn1 mutation is the most common class of mutation causing MarfanSyndrome. Typically, the aortic root in Fbn1C1039G mice undergoesprogressive dilatation, evident as early as 2 weeks of age. By 7 weeksof age, the aortic root in the mutant mice is significantly larger thanthat in wild-type mice. This observed size difference generally becomesmore pronounced over time. Histologic analysis of 14-week-old Fbn1C1039Gmice reveals aberrant thickening of the aortic media with fragmentationand disarray of elastic fibers. In addition, Fbn1C1039G mice showincreased collagen deposition, which is an indirect marker of increasedTGF-β signaling. Phosphorylation and subsequent nuclear translocation ofSmad2 (pSmad2) and Erk1/2 (pERK1/2), which are induced by TGF-βsignaling, are markedly increased in the aortic media of Fbn1C1039G micerelative to wild-type mice.

In order to determine the effects of angiotensin (1-7) peptides, hereTXA127, on the normal course of disease development, both wild-type andMarfan mice were each separated into two groups once the mice reached 2months of age. In each of the wild-type mice and Marfan mice, theplacebo control group received daily subcutaneous saline injections for60 days, while the TXA127 group received daily subcutaneous injectionsof TXA127 at a dose of 500 μg/kg/day for 60 days.

The primary endpoint of this study was degree of aortic growth observedover the 60 day treatment period, as measured by in vivoechocardiography. A baseline echocardiogram was performed just prior tothe beginning of the treatment period and then again just prior tosacrifice. After the 60 day treatment period, mice were sacrificed andtissue samples were taken for histological and other analysis.

As shown in FIG. 1, in Marfan mice, treatment with 500 μg/kg/day ofTXA127 resulted in a highly significant reduction in the degree ofaortic root growth as compared to Marfan mice treated with placebo. Infact, the data show that aortic root growth was essentially eliminatedin the TXA127 group. FIG. 1 also shows that treatment with TXA127 has nostatistically significant effect on the aortic root growth of wild typemice as compared to wild type mice treated with placebo.

This Example represents the first time that an Angiotensin (1-7) peptidehas been shown to have a significant effect on one of the most deadlysymptoms of Marfan Syndrome. Accordingly, several embodiments of thepresent invention provide a powerful and entirely new class oftreatments for Marfan sufferers.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

We claim:
 1. A method of treating Marfan Syndrome and/or a Marfan-related disorder comprising administering to a subject suffering from or susceptible to Marfan Syndrome and/or a Marfan-related disorder an angiotensin (1-7) peptide.
 2. The method of claim 1, wherein the angiotensin (1-7) peptide is administered at an effective dose periodically at an administration interval such that at least one symptom or feature of Marfan Syndrome and/or a Marfan-related disorder is reduced in intensity, severity, duration, or frequency or has delayed in onset.
 3. The method of claim 2, wherein the at least one symptom or feature of Marfan Syndrome and/or a Marfan-related disorder is selected from the group consisting of aortic enlargement, aortic dissection, eye lens dislocation, mitral valve prolapse, joint hypermobility, retinal detachment, strabismus, cataracts, glaucoma, obstructive lung disease, scoliosis, temporomandibular joint disorder, dural ectasia, and osteopenia.
 4. The method of claim 1, wherein the Marfan-related disorder is selected from the group consisting of: Loeys-Dietz Syndrome, Familial Aortic Aneurysm, Bicuspid Aortic Valve with Aortic Dilation, Familial Ectopia Lentis (dislocated lens), Mitral Valve Prolapse Syndrome, Marfan Habitus, Congenital Contractural Arachnodactyly (Beals Syndrome), Stickler syndrome, Shprintzen-Goldberg syndrome, Weill-Marchesani syndrome, and Ehlers-Danlos syndrome.
 5. The method of claim 1, wherein the angiotensin (1-7) peptide is administered parenterally.
 6. The method of claim 5, wherein the parenteral administration is selected from intravenous, intradermal, inhalation, transdermal (topical), intraocular, intramuscular, subcutaneous, intramuscular, and/or transmucosal administration.
 7. The method of claim 1, wherein the angiotensin (1-7) peptide is administered orally.
 8. The method of any of the preceding claims, wherein the angiotensin (1-7) peptide is administered monthly, weekly, daily, or at variable intervals.
 9. The method of any of the preceding claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 1-1,000 μg/kg/day.
 10. The method of any of the preceding claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 50-500 μg/kg/day.
 11. The method of any of the preceding claims, wherein the angiotensin (1-7) peptide is administered at an effective dose ranging from about 400-500 μg/kg/day.
 12. The method of any of the preceding claims, wherein the angiotensin (1-7) peptide is administered in combination with one or more Marfan Syndrome and/or a Marfan-related disorder medications.
 13. The method of claim 12, wherein the one or more Marfan Syndrome and/or a Marfan-related disorder medications is selected from the group consisting of beta blockers, calcium channel blockers, ACE inhibitors, angiotensin II receptor antagonists (e.g. losartan), anticoagulants, and combinations thereof.
 14. The method of claim 1, wherein the angiotensin (1-7) peptide comprises the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO:1).
 15. The method of claim 1, wherein the angiotensin (1-7) peptide is a functional equivalent of SEQ ID NO:1.
 16. The method of claim 15, wherein the functional equivalent is a linear peptide.
 17. The method of claim 16, wherein the linear peptide comprises a sequence that includes at least four amino acids from the seven amino acids that appear in the naturally-occurring Angiotensin (1-7), wherein the at least four amino acids maintain their relative positions as they appear in the naturally-occurring Angiotensin (1-7).
 18. The method of claim 16, wherein the linear peptide contains 4-25 amino acids.
 19. The method of claim 16, wherein the linear peptide is a fragment of the naturally-occurring Angiotensin (1-7).
 20. The method of claim 16, wherein the linear peptide contains amino acid substitutions, deletions and/or insertions in the naturally-occurring Angiotensin (1-7).
 21. The method of claim 20, wherein the linear peptide has an amino acid sequence of Asp¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:2).
 22. The method of claim 15, wherein the functional equivalent is a cyclic peptide.
 23. The method of claim 22, wherein the cyclic peptide comprises a linkage between amino acids.
 24. The method of claim 23, wherein the linkage is located at residues corresponding to positions Tyr⁴ and Pro⁷ in naturally-occurring Angiotensin (1-7).
 25. The method of claim 23, wherein the linkage is a thioether bridge.
 26. The method of claim 22, wherein the cyclic peptide comprises an amino acid sequence otherwise identical to the naturally-occurring Angiotensin (1-7) amino acid sequence of Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO:1).
 27. The method of claim 22, wherein the cyclic peptide is a 4,7-cyclized angiotensin (1-7) with the following formula:


28. The method of claim 15, wherein the angiotensin (1-7) peptide comprises one or more chemical modifications to increase protease resistance, serum stability and/or bioavailability.
 29. The method of claim 28, wherein the one or more chemical modifications comprise pegylation.
 30. A method of treating Marfan Syndrome and/or a Marfan-related disorder comprising administering to a subject who is suffering from or susceptible to Marfan Syndrome and/or a Marfan-related disorder an angiotensin (1-7) receptor agonist.
 31. The method of claim 30, wherein the angiotensin (1-7) receptor agonist is a non-peptidic agonist.
 32. The method of claim 31, wherein the non-peptidic agonist is a compound with the following structure:

or a pharmaceutically acceptable salt thereof.
 33. The method of any one of claims 30-32, wherein the angiotensin (1-7) receptor agonist is administered orally. 